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Thursday, August 31st, 2023 - Gonads

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Bayer, L. V., Milano, S., Formel, S. K., Kaur, H., Ravichandran, R., Cambeiro, J. A., Slinko, L., Catrina, I. E. and Bratu, D. P. (2023). Cup is essential for oskar mRNA translational repression during early Drosophila oogenesis. RNA Biol 20(1): 573-587. PubMed ID: 37553798
Summary:
Study of the timing and location for mRNA translation across model systems has begun to shed light on molecular events fundamental to such processes as intercellular communication, morphogenesis, and body pattern formation. In D. melanogaster, the posterior mRNA determinant, oskar, is transcribed maternally but translated only when properly localized at the oocyte's posterior cortex. Two effector proteins, Bruno1 and Cup, mediate steps of oskar mRNA regulation. The current model in the field identifies Bruno1 as necessary for Cup's recruitment to oskar mRNA and indispensable for oskar's translational repression. This study now report that this Bruno1-Cup interaction leads to precise oskar mRNA regulation during early oogenesis and, importantly, the two proteins mutually influence each other's mRNA expression and protein distribution in the egg chamber. These factors were shown to be stably associated with oskar mRNA in vivo. Cup associates with oskar mRNA without Bruno1, while surprisingly Bruno1's stable association with oskar mRNA depends on Cup. The essential factor for oskar mRNA repression in early oogenesis is Cup, not Bruno1. Furthermore, it was found that Cup is a key P-body component that maintains functional P-body morphology during oogenesis and is necessary for oskar mRNA's association with P-bodies. Therefore, Cup drives the translational repression and stability of oskar mRNA. These experimental results point to a regulatory feedback loop between Bruno 1 and Cup in early oogenesis that appears crucial for oskar mRNA to reach the posterior pole and its expression in the egg chamber for accurate embryo development.
Bailly, T. P. M., Kohlmeier, P., Etienne, R. S., Wertheim, B. and Billeter, J. C. (2023). Social modulation of oogenesis and egg laying in Drosophila melanogaster. Curr Biol 33(14): 2865-2877.e2864. PubMed ID: 37369209
Summary:
Being part of a group facilitates cooperation between group members but also creates competition for resources. This is a conundrum for gravid females, whose future offspring benefit from being in a group only if there are enough resources relative to group size. Females may therefore be expected to modulate reproductive output depending on social context. In the fruit fly Drosophila melanogaster, females actively attract conspecifics to lay eggs on the same resources, generating groups in which individuals may cooperate or compete. The genetic tractability of this species allows dissecting the mechanisms underlying physiological adaptation to social context. This study shows that females produce eggs increasingly faster as group size increases. By laying eggs faster when grouped than when isolated, females reduce competition between offspring and increase offspring survival. In addition, grouped females lay eggs during the day, while isolated females lay them at night. This study shows that responses to the presence of others requires visual input and that flies from any sex, mating status, or species can trigger these responses. The mechanisms of this modulation of egg laying by group is connected to a lifting of the inhibition of light on oogenesis and egg laying, possibly mediated in part by an increase in juvenile hormone activity. Because modulation of reproduction by social context is a hallmark of animals with higher levels of sociality, these findings in a species considered solitary question the validity of this nomenclature and suggest a widespread and profound influence of social context on reproduction.
Butsch, T. J., Dubuisson, O., Johnson, A. E. and Bohnert, K. A. (2023). VCP promotes tTAF-target gene expression and spermatocyte differentiation by downregulating mono-ubiquitylated H2A. Development 150(14). PubMed ID: 37401420
Summary:
Valosin-containing protein (VCP) binds and extracts ubiquitylated cargo to regulate protein homeostasis. VCP has been studied primarily in aging and disease contexts, but it also affects germline development. However, the precise molecular functions of VCP in the germline, particularly in males, are poorly understood. Using the Drosophila male germline as a model system, it was found that VCP translocates from the cytosol to the nucleus as germ cells transition into the meiotic spermatocyte stage. Importantly, nuclear translocation of VCP appears to be one crucial event stimulated by testis-specific TBP-associated factors (tTAFs) to drive spermatocyte differentiation. VCP promotes the expression of several tTAF-target genes, and VCP knockdown, like tTAF loss of function, causes cells to arrest in early meiotic stages. At a molecular level, VCP activity supports spermatocyte gene expression by downregulating a repressive histone modification, mono-ubiquitylated H2A (H2Aub), during meiosis. Remarkably, experimentally blocking H2Aub in VCP-RNAi testes is sufficient to overcome the meiotic-arrest phenotype and to promote development through the spermatocyte stage. Collectively, these data highlight VCP as a downstream effector of tTAFs that downregulates H2Aub to facilitate meiotic progression.
Yu, J., Fu, Y., Li, Z., Huang, Q., Tang, J., Sun, C., Zhou, P., He, L., Sun, F., Cheng, X., Ji, L., Yu, H., Shi, Y., Gu, Z., Sun, F. and Zhao, X. (2023). Single-cell RNA sequencing reveals cell landscape following antimony exposure during spermatogenesis in Drosophila testes. Cell Death Discov 9(1): 86. PubMed ID: 36894529
Summary:
Antimony (Sb), is thought to induce testicular toxicity, although this remains controversial. This study investigated the effects of Sb exposure during spermatogenesis in the Drosophila testis and the underlying transcriptional regulatory mechanism at single-cell resolution. Firstly, it was found that flies exposed to Sb for 10 days led to dose-dependent reproductive toxicity during spermatogenesis. Protein expression and RNA levels were measured by immunofluorescence and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) was performed to characterize testicular cell composition and identify the transcriptional regulatory network after Sb exposure in Drosophila testes. scRNA-seq analysis revealed that Sb exposure influenced various testicular cell populations, especially in GSCs_to_Early_Spermatogonia and Spermatids clusters. Importantly, carbon metabolism was involved in GSCs/early spermatogonia maintenance and positively related with SCP-Containing Proteins, S-LAPs, and Mst84D signatures. Moreover, Seminal Fluid Proteins, Mst57D, and Serpin signatures were highly positively correlated with spermatid maturation. Pseudotime trajectory analysis revealed three novel states for the complexity of germ cell differentiation, and many novel genes (e.g., Dup98B) were found to be expressed in state-biased manners during spermatogenesis. Collectively, this study indicates that Sb exposure negatively impacts GSC maintenance and spermatid elongation, damaging spermatogenesis homeostasis via multiple signatures in Drosophila testes and therefore supporting Sb-mediated testicular toxicity.
Zhang, Q., Zhang, Y., Zhang, Q., Li, L. and Zhao, S. (2023). Division Promotes Adult Stem Cells to Perform Active Niche Competition. Genetics. PubMed ID: 36892331
Summary:
Adult stem cells maintain homeostatic self-renewal through the strategy of either population or single-cell asymmetry, and the former type of stem cells are thought to take passive while the latter ones take active competition for niche occupancy. Although the division ability of stem cells is known to be crucial for their passive competition, whether it is also crucial for active competition is still elusive. Drosophila female germline stem cells are thought to take active competition, and bam mutant germ cells are more competitive than wild-type germline stem cells for niche occupancy. This study reports that either cycB, cycE, cdk2, or rheb null mutation drastically attenuates the division ability and niche-occupancy capacity of bam mutant germ cells. Conversely, accelerating their cell cycle by mutating hpo has an enhanced effect. Last but not least, it was also determined that E-Cadherin, which was proposed to be crucial previously, just plays a mild role in bam mutant germline niche occupancy. Together with previous studies, it is proposed that division ability plays a unified crucial role in either active or passive competition among stem cells for niche occupancy.
Xing, Y., Larson, K., Li, J. and Li, W. X. (2023). Canonical and non-canonical functions of STAT in germline stem cell maintenance. Dev Dyn. PubMed ID: 36866634
Summary:
Maintenance of the Drosophila male germline stem cells (GSCs) requires activation of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway by niche signals. The precise role of JAK/STAT signaling in GSC maintenance, however, remains incompletely understood. This study shows that, GSC maintenance requires both canonical and non-canonical JAK/STAT signaling, in which unphosphorylated STAT (uSTAT) maintains heterochromatin stability by binding to heterochromatin protein 1 (HP1). GSC-specific overexpressing STAT, or even the transcriptionally inactive mutant STAT, increases GSC number and partially rescues the GSC-loss mutant phenotype due to reduced JAK activity. Furthermore, both HP1 and STAT were found to be transcriptional targets of the canonical JAK/STAT pathway in GSCs, and that GSCs exhibit higher heterochromatin content. These results suggest that persistent JAK/STAT activation by niche signals leads to the accumulation of HP1 and uSTAT in GSCs, which promote heterochromatin formation important for maintaining GSC identity. Thus, the maintenance of Drosophila GSCs requires both canonical and non-canonical STAT functions within GSCs for heterochromatin regulation.

Wednesday, August 30th - Disease Models

Vourkou, E., Rouiz Ortega, E. D., Mahajan, S., Mudher, A. and Skoulakis, E. M. C. (2023). Human Tau Aggregates Are Permissive to Protein Synthesis-Dependent Memory in Drosophila Tauopathy Models. J Neurosci 43(16): 2988-3006. PubMed ID: 36868851
Summary:
Tauopathies including Alzheimer's disease, are characterized by progressive cognitive decline, neurodegeneration, and intraneuronal aggregates comprised largely of the axonal protein Tau. It has been unclear whether cognitive deficits are a consequence of aggregate accumulation thought to compromise neuronal health and eventually lead to neurodegeneration. This study use the Drosophila tauopathy model and mixed-sex populations to reveal an adult onset pan-neuronal Tau accumulation-dependent decline in learning efficacy and a specific defect in protein synthesis-dependent memory (PSD-M), but not in its protein synthesis-independent variant. It was demonstrated that these neuroplasticity defects are reversible on suppression of new transgenic human Tau expression and surprisingly correlate with an increase in Tau aggregates. Inhibition of aggregate formation via acute oral administration of methylene blue results in re-emergence of deficient memory in animals with suppressed human Tau (hTau)(0N4R) expression. Significantly, aggregate inhibition results in PSD-M deficits in hTau(0N3R)-expressing animals, which present elevated aggregates and normal memory if untreated with methylene blue. Moreover, methylene blue-dependent hTau(0N4R) aggregate suppression within adult mushroom body neurons also resulted in emergence of memory deficits. Therefore, deficient PSD-M on human Tau expression in the Drosophila CNS is not a consequence of toxicity and neuronal loss because it is reversible. Furthermore, PSD-M deficits do not result from aggregate accumulation, which appears permissive, if not protective of processes underlying this memory variant.
Veselkina, E. R., Trostnikov, M. V., Roshina, N. V. and Pasyukova, E. G. (2023). The Effect of the Tau Protein on D. melanogaster Lifespan Depends on GSK3 Expression and Sex. Int J Mol Sci 24(3). PubMed ID: 36768490
Summary:
The microtubule-associated conserved protein tau has attracted significant attention because of its essential role in the formation of pathological changes in the nervous system, which can reduce longevity. The study of the effects caused by tau dysfunction and the molecular mechanisms underlying them is complicated because different forms of tau exist in humans and model organisms, and the changes in protein expression can be multidirectional. This article shows that an increase in the expression of the main isoform of the Drosophila melanogaster Tau protein in the nervous system has differing effects on lifespan depending on the sex of individuals but has no effect on the properties of the nervous system, in particular, the synaptic activity and distribution of another microtubule-associated protein, Futsch, in neuromuscular junctions. Reduced expression of tau in the nervous system does not affect the lifespan of wild-type flies, but it does increase the lifespan dramatically shortened by overexpression of the shaggy gene encoding the GSK3 (Glycogen Synthase Kinase 3) protein kinase, which is one of the key regulators of tau phosphorylation levels. This effect is accompanied by the normalization of the Futsch protein distribution impaired by shaggy overexpression. The results presented in this article demonstrate that multidirectional changes in tau expression can lead to effects that depend on the sex of individuals and the expression level of GSK3.
Xie, S., Xu, C., Wu, C., Lou, Y., Duan, J., Sang, R., Lou, Z., Hou, J., Ge, W., Xi, Y. and Yang, X. (2023). Co-dependent regulation of p-BRAF and potassium channel KCNMA1 levels drives glioma progression. Cell Mol Life Sci 80(3): 61. PubMed ID: 36763212
Summary:
BRAF mutations have been found in gliomas which exhibit abnormal electrophysiological activities, implying their potential links with the ion channel functions. This study identified the Drosophila potassium channel, Slowpoke (Slo), the ortholog of human KCNMA1, as a critical factor involved in dRafGOF glioma progression. Slo was upregulated in dRafGOF glioma. Knockdown of slo led to decreases in dRafGOF levels, glioma cell proliferation, and tumor-related phenotypes. Overexpression of slo in glial cells elevated dRaf expression and promoted cell proliferation. Similar mutual regulations of p-BRAF and KCNMA1 levels were then recapitulated in human glioma cells with the BRAF mutation. Elevated p-BRAF and KCNMA1 were also observed in HEK293T cells upon the treatment of 20 mM KCl, which causes membrane depolarization. Knockdown KCNMA1 in these cells led to a further decrease in cell viability. Based on these results, it is concluded that the levels of p-BRAF and KCNMA1 are co-dependent and mutually regulated. It is proposed that, in depolarized glioma cells with BRAF mutations, high KCNMA1 levels act to repolarize membrane potential and facilitate cell growth. This study provides a new strategy to antagonize the progression of gliomas as induced by BRAF mutations.
Adedayo, B. C., Akinniyi, S. T., Ogunsuyi, O. B. and Oboh, G. (2023). In the quest for the ideal sweetener: Aspartame exacerbates selected biomarkers in the fruit fly (Drosophila melanogaster) model of Alzheimer's disease more than sucrose. Aging Brain 4: 100090. PubMed ID: 37559954
Summary:
This study evaluated the effect of dietary inclusions of aspartame and sucrose on some selected behavioral and biochemical indices linked with Alzheimer's disease in a transgenic fruit fly (Drosophila melanogaster) model expressing human amyloid precursor protein and secretase. Flies were raised on a diet supplemented with sucrose and aspartame for 14 days. Thereafter, the flies were assessed for their survival rate, learning and memory, as well as locomotor performance, 14 days post-treatment. This was followed by homogenising the fly heads, and the homogenates were assayed for acetylcholinesterase and monoamine oxidase activities, as well as levels of lipid peroxidation, reactive oxygen species, and total thiol. The results showed aspartame at all levels of dietary intake and a high proportion of sucrose significantly aggravated the mortality rate, locomotor deficiency, and impaired biomarkers of oxidative stress and antioxidant status in the transgenic flies, while no significant effect was found on acetylcholinesterase activity or memory function. These findings therefore suggest that while low dietary inclusions of sucrose are tolerable under AD-like phenotypes in the flies, high inclusions of sucrose and all proportions of aspartame tested aggravated mortality rate, locomotion and oxidative stress in the flies.
Barwell, T., Raina, S., Page, A., MacCharles, H. and Seroude, L. (2023). Juvenile and adult expression of polyglutamine expanded huntingtin produce distinct aggregate distributions in Drosophila muscle. Hum Mol Genet 32(16): 2656-2668. PubMed ID: 37369041
Summary:
While Huntington's disease (HD) is widely recognized as a disease affecting the nervous system, much evidence has accumulated to suggest peripheral or non-neuronal tissues are affected as well. This study utilized the UAS/GAL4 system to express a pathogenic HD construct in the muscle of the fly and characterize the effects. Detrimental phenotypes were observed such as a reduced lifespan, decreased locomotion and accumulation of protein aggregates. Strikingly, depending on the GAL4 driver used to express the construct, different aggregate distributions and severity of phenotypes were seen. These different aggregate distributions were found to be dependent on the expression level and the timing of expression. Hsp70, a well-documented suppressor of polyglutamine aggregates, was found to strongly reduce the accumulation of aggregates in the eye, but in the muscle, it did not prevent the reduction of the lifespan. Therefore, the molecular mechanisms underlying the detrimental effects of aggregates in the muscle are distinct from the nervous system.
Ayers, K. L., Eggers, S., ..., BenZeev, B. and Sinclair, A. H. (2023). Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects. Nat Commun 14(1): 3403. PubMed ID: 37296101
Summary:
Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Thus study identified recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 (Rnp4F) reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which is tentatively proposed to be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). These findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Tuesday, August 29th - Behavior

Adams, G. J. and O'Brien, P. A. (2023). The unified theory of sleep: Eukaryotes endosymbiotic relationship with mitochondria and REM the push-back response for awakening. Neurobiol Sleep Circadian Rhythms 15: 100100. PubMed ID: 37484687
Summary:
The Unified Theory suggests that sleep is a process that developed in eukaryotic animals from a relationship with an endosymbiotic bacterium. Over evolutionary time the bacterium evolved into the modern mitochondrion that continues to exert an effect on sleep patterns, e.g. the bacterium Wolbachia establishes an endosymbiotic relationship with Drosophila and many other species of insects and is able to change the host's behaviour by making it sleep. The hypothesis is supported by other host-parasite relationships, e.g., Trypanosoma brucei which causes day-time sleepiness and night-time insomnia in humans and cattle. For eukaryotes such as Monocercomonoids that don't contain mitochondria, no evidence was found of them sleeping. Mitochondria produce the neurotransmitter gamma aminobutyric acid (GABA), and ornithine a precursor of the neurotransmitter GABA, together with substances such as 3,4dihydroxy phenylalanine (DOPA) a precursor for the neurotransmitter dopamine: These substances have been shown to affect the sleep/wake cycles in animals such as Drosophilia and Hydra. Eukaryote animals have traded the very positive side of having mitochondria providing aerobic respiration for them with the negative side of having to sleep. NREM (Quiet sleep) is the process endosymbionts have imposed upon their host eukaryotes and REM (Active sleep) is the push-back adaptation of eukaryotes with brains, returning to wakefulness.
Troup, M., Tainton-Heap, L. A. L. and van Swinderen, B. (2023). Neural Ensemble Fragmentation in the Anesthetized Drosophila Brain. J Neurosci 43(14): 2537-2551. PubMed ID: 36868857
Summary:
General anesthetics cause a profound loss of behavioral responsiveness in all animals. In mammals, general anesthesia is induced in part by the potentiation of endogenous sleep-promoting circuits, although "deep" anesthesia is understood to be more similar to coma. Surgically relevant concentrations of anesthetics, such as isoflurane and propofol, have been shown to impair neural connectivity across the mammalian brain, which presents one explanation why animals become largely unresponsive when exposed to these drugs. It remains unclear whether general anesthetics affect brain dynamics similarly in all animal brains, or whether simpler animals, such as insects, even display levels of neural connectivity that could be disrupted by these drugs. This study used whole-brain calcium imaging in behaving female Drosophila flies to investigate whether isoflurane anesthesia induction activates sleep-promoting neurons, and then inquired how all other neurons across the fly brain behave under sustained anesthesia. It was possible to track the activity of hundreds of neurons simultaneously during waking and anesthetized states, for spontaneous conditions as well as in response to visual and mechanical stimuli. Whole-brain dynamics and connectivity were compared under isoflurane exposure to optogenetically induced sleep. Neurons in the Drosophila brain remain active during general anesthesia as well as induced sleep, although flies become behaviorally inert under both treatments. This study identified surprisingly dynamic neural correlation patterns in the waking fly brain, suggesting ensemble-like behavior. These become more fragmented and less diverse under anesthesia but remain wake-like during induced sleep.
Wice, E. W. and Saltz, J. B. (2023). Indirect genetic effects for social network structure in Drosophila melanogaster. Philos Trans R Soc Lond B Biol Sci 378(1874): 20220075. PubMed ID: 36802774
Summary:
The position an individual holds in a social network is dependent on both its direct and indirect social interactions. Because social network position is dependent on the actions and interactions of conspecifics, it is likely that the genotypic composition of individuals within a social group impacts individuals' network positions. However, little is known about whether social network positions have a genetic basis, and even less about how the genotypic makeup of a social group impacts network positions and structure. With ample evidence indicating that network positions influence various fitness metrics, studying how direct and indirect genetic effects shape network positions is crucial for furthering understanding of how the social environment can respond to selection and evolve. Using replicate genotypes of Drosophila melanogaster fruit flies, social groups were created that varied in their genotypic makeup. Social groups were videoed, and networks were generated using motion-tracking software. It was found that both an individual's own genotype and the genotypes of conspecifics in its social group affect its position within a social network. These findings provide an early example of how indirect genetic effects and social network theory can be linked, and shed new light on how quantitative genetic variation shapes the structure of social groups.
Amanullah, A., Arzoo, S., Aslam, A., Qureshi, I. W. and Hussain, M. (2023). Inbreeding-Driven Innate Behavioral Changes in Drosophila melanogaster. Biology (Basel) 12(7). PubMed ID: 37508357
Summary:
Drosophila melanogaster has long been used to demonstrate the effect of inbreeding, particularly in relation to reproductive fitness and stress tolerance. In comparison, less attention has been given to exploring the influence of inbreeding on the innate behavior of D. melanogaster. In this study, multiple replicates of six different types of crosses were set in pair conformation of the laboratory-maintained wild-type D. melanogaster. This resulted in progeny with six different levels of inbreeding coefficients. Larvae and adult flies of varied inbreeding coefficients were subjected to different behavioral assays. In addition to the expected inbreeding depression in the-egg to-adult viability, noticeable aberrations were observed in the crawling and phototaxis behaviors of larvae. Negative geotactic behavior as well as positive phototactic behavior of the flies were also found to be adversely affected with increasing levels of inbreeding. Interestingly, positively phototactic inbred flies demonstrated improved learning compared to outbred flies, potentially the consequence of purging. Flies with higher levels of inbreeding exhibited a delay in the manifestation of aggression and courtship. In summary, these findings demonstrate that inbreeding influences the innate behaviors in D. melanogaster, which in turn may affect the overall biological fitness of the flies.
Yang, Y. T., Hu, S. W., Li, X., Sun, Y., He, P., Kohlmeier, K. A. and Zhu, Y. (2023). Sex peptide regulates female receptivity through serotoninergic neurons in Drosophila. iScience 26(3): 106123. PubMed ID: 36876123
Summary:
The courtship ritual is a dynamic interplay between males and females. Courtship successfully leading to copulation is determined by the intention of both parties which is conveyed by complex action sequences. In Drosophila, the neural mechanisms controlling the female's willingness to mate, or sexual receptivity, have only recently become the focus of investigations. This study reports that pre-mating sexual receptivity in females requires activity within a subset of serotonergic projection neurons (SPNs), which positively regulate courtship success. Of interest, a male-derived sex peptide, SP, which was transferred to females during copulation acted to inhibit the activity of SPN and suppressed receptivity. Downstream of 5-HT, subsets of 5-HT7 receptor neurons played critical roles in SP-induced suppression of sexual receptivity. Together, this study reveals a complex serotonin signaling system in the central brain of Drosophila which manages the female's desire to mate.
Xu, L., Jiang, H. B., Yu, J. L., Pan, D., Tao, Y., Lei, Q., Chen, Y., Liu, Z. and Wang, J. J. (2023). Two odorant receptors regulate 1-octen-3-ol induced oviposition behavior in the oriental fruit fly. Commun Biol 6(1): 176. PubMed ID: 36792777
Summary:
The oriental fruit fly Bactrocera dorsalis (Hendel) is a notorious pest of fruit crops. Gravid females locate suitable oviposition sites by detecting host plant volatiles. This study demonstrates that 1-octen-3-ol, a volatile from mango, guides the oviposition behavior of female flies. Two odorant receptors (BdorOR7a-6 and BdorOR13a) are identified as key receptors for 1-octen-3-ol perception by qPCR analysis, heterologous expression in Xenopus laevis oocytes and HEK 293 cells followed by in vitro binding assays, as well as CRISPR/Cas9 genome editing in B. dorsalis. Molecular docking and site-directed mutagenesis are used to determine major binding sites for 1-octen-3-ol. The results demonstrate the potential of 1-octen-3-ol to attract gravid females and molecular mechanism of its perception in B. dorsalis. BdorOR7a-6 and BdorOR13a can therefore be used as molecular targets for the development of female attractants. Furthermore, site-directed mutagenesis data will facilitate the chemical engineering of 1-octen-3-ol to generate more efficient attractants.

Monday, August 28th - Signaling

Zhang, W., Wang, D., Si, J., Jin, L. H. and Hao, Y. (2023). Gbb Regulates Blood Cell Proliferation and Differentiation through JNK and EGFR Signaling Pathways in the Drosophila Lymph Gland. Cells 12(4). PubMed ID: 36831328
Summary:
The Drosophila lymph gland is an ideal model for studying hematopoiesis, and unraveling the mechanisms of Drosophila hematopoiesis can improve understanding of the pathogenesis of human hematopoietic malignancies. Bone morphogenetic protein (BMP) signaling is involved in a variety of biological processes and is highly conserved between Drosophila and mammals. Decapentaplegic (Dpp)/BMP signaling is known to limit posterior signaling center (PSC) cell proliferation by repressing the protooncogene dmyc. However, the role of two other TGF-β family ligands, Glass bottom boat (Gbb) and Screw (Scw), in Drosophila hematopoiesis is currently largely unknown. This study showed that the loss of Gbb in the cortical zone (CZ) induced lamellocyte differentiation by overactivation of the EGFR and JNK pathways and caused excessive differentiation of plasmatocytes, mainly by the hyperactivation of EGFR. Furthermore, it was found that Gbb was also required for preventing the hyperproliferation of the lymph glands by inhibiting the overactivation of the Epidermal Growth Factor Receptor (EGFR) and c-Jun N-terminal Kinase (JNK) pathways. These results further advance understanding of the roles of Gbb protein and the BMP signaling in Drosophila hematopoiesis and the regulatory relationship between the BMP, EGFR, and JNK pathways in the proliferation and differentiation of lymph gland hemocytes.
Clements, R., Smith, T., Cowart, L., Zhumi, J., Sherrod, A., Cahill, A. and Hunter, G. L. (2023). Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition. bioRxiv. PubMed ID: 37461640
Summary:
The self-organization of cells during development is essential for the formation of healthy tissues, and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. This study investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. Myosin XV was shown to be required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. It is concluded that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV is present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.
Bare, Y., Matusek, T., Vriz, S., Deffieu, M. S., Therond, P. P. and Gaudin, R. (2023). TMED10 mediates the loading of neosynthesised Sonic Hedgehog in COPII vesicles for efficient secretion and signalling. Cell Mol Life Sci 80(9): 266. PubMed ID: 37624561
Summary:
The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. This study employed the retention using selective hooks (RUSH) system to show that newly-synthesised SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, the Drosophila wing imaginal disc model was used to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, this work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesised SHH secretion with potential impact on embryonic development.
Yoshida, K. and Hayashi, S. (2023). Epidermal growth factor receptor signaling protects epithelia from morphogenetic instability and tissue damage in Drosophila. Development 150(5). PubMed ID: 36897356
Summary:
Dying cells in the epithelia communicate with neighboring cells to initiate coordinated cell removal to maintain epithelial integrity. Naturally occurring apoptotic cells are mostly extruded basally and engulfed by macrophages. This study has investigated the role of Epidermal growth factor (EGF) receptor (EGFR) signaling in the maintenance of epithelial homeostasis. In Drosophila embryos, epithelial tissues undergoing groove formation preferentially enhanced extracellular signal-regulated kinase (ERK) signaling. In EGFR mutant embryos at stage 11, sporadic apical cell extrusion in the head initiates a cascade of apical extrusions of apoptotic and non-apoptotic cells that sweeps the entire ventral body wall. This study shows that this process is apoptosis dependent, and clustered apoptosis, groove formation, and wounding sensitize EGFR mutant epithelia to initiate massive tissue disintegration. It was further shown that tissue detachment from the vitelline membrane, which frequently occurs during morphogenetic processes, is a key trigger for the EGFR mutant phenotype. These findings indicate that, in addition to cell survival, EGFR plays a role in maintaining epithelial integrity, which is essential for protecting tissues from transient instability caused by morphogenetic movement and damage.
Zhao, H., Moberg, K. H. and Veraksa, A. (2023). Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye. Dev Cell 58(5): 416-434.e412. PubMed ID: 36868234
Summary:
The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. This study identified a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. This work expands the range of functions and regulatory mechanisms under Hippo pathway control.
Zhou, J., He, L., Liu, M., Guo, X., Du, G., Yan, L., Zhang, Z., Zhong, Z. and Chen, H. (2023). Sleep loss impairs intestinal stem cell function and gut homeostasis through the modulation of the GABA signalling pathway in Drosophila. Cell Prolif: e13437. PubMed ID: 36869584
Summary:
Sleep is essential for maintaining health. Indeed, sleep loss is closely associated with multiple health problems, including gastrointestinal disorders. However, it is not yet clear whether sleep loss affects the function of intestinal stem cells (ISCs). Mechanical sleep deprivation and sss mutant flies were used to generate the sleep loss model. qRT-PCR was used to measure the relative mRNA expression. Gene knock-in flies were used to observe protein localization and expression patterns. Immunofluorescence staining was used to determine the intestinal phenotype. The shift in gut microbiota was observed using 16S rRNA sequencing and analysis. Sleep loss caused by mechanical sleep deprivation and sss mutants disturbs ISC proliferation and intestinal epithelial repair through the brain-gut axis. In addition, disruption of SSS causes gut microbiota dysbiosis in Drosophila. As regards the mechanism, gut microbiota and the GABA signalling pathway both partially played a role in the sss regulation of ISC proliferation and gut function. The research shows that sleep loss disturbed ISC proliferation, gut microbiota, and gut function. Therefore, the results offer a stem cell perspective on brain-gut communication, with details on the effect of the environment on ISCs.

Friday, August 25th - Adult Neural Development and Function

Qie, X., Ren, Y., Chen, X., Du, Y., Dong, K. and Hu, Z. (2023). Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A. Insect Sci. PubMed ID: 36752392
Summary:
Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation of stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and HA was found to have excellent insecticidal activity and is worthy of further study as a botanical insecticide. This study performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w1118 (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1 DSC1-/-, electrophysiology results implicated that HA delayed the decay rate and increased the frequency of miniature excitatory junctional potentials of Drosophila from w(1118) , para(ts1) , and DSC1-/- strains. Moreover, the neuromuscular synapse excitatory activities of parats1DSC1-/- larvae were more sensitive to HA than DSC1-/- larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, this study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya.
Osaka, J., Yasuda, H., Watanuki, Y., Kato, Y., Nitta, Y., Sugie, A., Sato, M. and Suzuki, T. (2023). Identification of genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. Genes Genet Syst 97(6): 297-309. PubMed ID: 36878557
Summary:
Neural activity-dependent synaptic plasticity is an important physiological phenomenon underlying environmental adaptation, memory and learning. However, its molecular basis, especially in presynaptic neurons, is not well understood. Previous studies have shown that the number of presynaptic active zones in the Drosophila melanogaster photoreceptor R8 is reversibly changed in an activity-dependent manner. During reversible synaptic changes, both synaptic disassembly and assembly processes were observed. Although this study has established a paradigm for screening molecules involved in synaptic stability and several genes have been identified, genes involved in stimulus-dependent synaptic assembly are still elusive. Therefore, the aim of this study was to identify genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. To this end, RNAi screening was performed against 300 memory-defective, synapse-related or transmembrane molecules in photoreceptor R8 neurons. Candidate genes were narrowed down to 27 genes in the first screen using presynaptic protein aggregation as a sign of synaptic disassembly. In the second screen, the decreasing synapse number was directly quantified using a GFP-tagged presynaptic protein marker. Custom-made image analysis software was used, which automatically locates synapses and counts their number along individual R8 axons, and identified cirl was used as a candidate gene responsible for synaptic assembly. Finally, a new model is presented of stimulus-dependent synaptic assembly through the interaction of cirl and its possible ligand, ten-a. This study demonstrates the feasibility of using the automated synapse quantification system to explore activity-dependent synaptic plasticity in Drosophila R8 photoreceptors in order to identify molecules involved in stimulus-dependent synaptic assembly.
Pan, G., Li, R., Xu, G., Weng, S., Yang, X. L., Yang, L. and Ye, B. (2023). Cross-modal modulation gates nociceptive inputs in Drosophila. Curr Biol 33(7): 1372-1380. PubMed ID: 36893758
Summary:
Animals' response to a stimulus in one sensory modality is usually influenced by other modalities. One important type of multisensory integration is the cross-modal modulation, in which one sensory modality modulates (typically inhibits) another. Identification of the mechanisms underlying cross-modal modulations is crucial for understanding how sensory inputs shape animals' perception and for understanding sensory processing disorders. However, the synaptic and circuit mechanisms that underlie cross-modal modulation are poorly understood. This is due to the difficulty of separating cross-modal modulation from multisensory integrations in neurons that receive excitatory inputs from two or more sensory modalities(5)-in which case it is unclear what the modulating or modulated modality is. This study reports a unique system for studying cross-modal modulation by taking advantage of the genetic resources in Drosophila. Gentle mechanical stimuli was shown to inhibit nociceptive responses in Drosophila larvae. Low-threshold mechanosensory neurons inhibit a key second-order neuron in the nociceptive pathway through metabotropic GABA receptors on nociceptor synaptic terminals. Strikingly, this cross-modal inhibition is only effective when nociceptor inputs are weak, thus serving as a gating mechanism for filtering out weak nociceptive inputs. These findings unveil a novel cross-modal gating mechanism for sensory pathways.
Meissner, G. W., Nern, A., Dorman, Z., DePasquale, G. M., Forster, K., Gibney, T., Hausenfluck, J. H., He, Y., Iyer, N. A., Jeter, J., Johnson, L., Johnston, R. M., Lee, K., Melton, B., Yarbrough, B., Zugates, C. T., Clements, J., Goina, C., Otsuna, H., Rokicki, K., Svirskas, R. R., Aso, Y., Card, G. M., Dickson, B. J., Ehrhardt, E., Goldammer, J., Ito, M., Kainmueller, D., Korff, W., Mais, L., Minegishi, R., Namiki, S., Rubin, G. M., Sterne, G. R., Wolff, T. and Malkesman, O. (2023). A searchable image resource of Drosophila GAL4 driver expression patterns with single neuron resolution. Elife 12. PubMed ID: 36820523
Summary:
Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. This study used stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. Aligned images of 74,000 such adult central nervous systems is being released. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, the images have been made searchable on the NeuronBridge website. This study demonstrates the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.
Winding, M., Pedigo, B. D., Barnes, C. L., Patsolic, H. G., Park, Y., Kazimiers, T., Fushiki, A., Andrade, I. V., Khandelwal, A., Valdes-Aleman, J., Li, F., Randel, N., Barsotti, E., Correia, A., Fetter, R. D., Hartenstein, V., Priebe, C. E., Vogelstein, J. T., Cardona, A. and Zlatic, M. (2023). The connectome of an insect brain. Science 379(6636): eadd9330. PubMed ID: 36893230
Summary:
Brains contain networks of interconnected neurons and so knowing the network architecture is essential for understanding brain function. This study therefore mapped the synaptic-resolution connectome of an entire insect brain (Drosophila larva) with rich behavior, including learning, value computation, and action selection, comprising 3016 neurons and 548,000 synapses.This study characterized neuron types, hubs, feedforward and feedback pathways, as well as cross-hemisphere and brain-nerve cord interactions.Pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs were found. The brain's most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled state-of-the-art deep learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.
Zhang, Y., Lowe, S., Ding, A. Z. and Li, X. (2023).. Notch-dependent binary fate choice regulates the Netrin pathway to control axon guidance of Drosophila visual projection neurons. Cell Rep 42(3): 112143. PubMed ID: 36821442
Summary:
Notch-dependent binary fate choice between sister neurons is one of the mechanisms to generate neural diversity. How these upstream neural fate specification programs regulate downstream effector genes to control axon targeting and neuropil assembly remains less well understood. This tudy reports that Notch-dependent binary fate choice in Drosophila medulla neurons is required to regulate the Netrin axon guidance pathway, which controls targeting of transmedullary (Tm) neurons to lobula. In medulla neurons of Notch-on hemilineage composed of mostly lobula-targeting neurons, Notch signaling is required to activate the expression of Netrin-B and repress the expression of its repulsive receptor Unc-5. Turning off Unc-5 is necessary for Tm neurons to target lobula. Furthermore, Netrin-B provided by Notch-on medulla neurons is required for correct targeting of Tm axons from later-generated medulla columns. Thus, the coordinate regulation of Netrin pathway components by Notch signaling ensures correct targeting of Tm axons and contributes to the neuropil assembly.

Thursday, August 24th - Enhancers and Transcriptional Regulation

Raja, K. K. B., Bachman, E. A., Fernholz, C. E., Trine, D. S., Hobmeier, R. E., Maki, N. J., Massoglia, T. J. and Werner, T. (2023). The Genetic Mechanisms Underlying the Concerted Expression of the yellow and tan Genes in Complex Patterns on the Abdomen and Wings of Drosophila guttifera. Genes (Basel) 14(2). PubMed ID: 36833231
Summary:
How complex morphological patterns form is an intriguing question in developmental biology. However, the mechanisms that generate complex patterns remain largely unknown. This study sought to identify the genetic mechanisms that regulate the tan (t) gene in a multi-spotted pigmentation pattern on the abdomen and wings of Drosophila guttifera. Previously, it was shown that yellow (y) gene expression completely prefigures the abdominal and wing pigment patterns of this species. In the current study, it was demonstrated that the t gene is co-expressed with the y gene in nearly identical patterns, both transcripts foreshadowing the adult abdominal and wing melanin spot patterns. Cis-regulatory modules (CRMs) were idnetified of t, one of which drives reporter expression in six longitudinal rows of spots on the developing pupal abdomen, while the second CRM activates the reporter gene in a spotted wing pattern. Comparing the abdominal spot CRMs of y and t, a similar composition of putative transcription factor binding sites was found that are thought to regulate the complex expression patterns of both terminal pigmentation genes y and t. In contrast, the y and t wing spots appear to be regulated by distinct upstream factors. The results suggest that the D. guttifera abdominal and wing melanin spot patterns have been established through the co-regulation of y and t, shedding light on how complex morphological traits may be regulated through the parallel coordination of downstream target genes.
Tabuloc, C. A., Cai, Y. D., Kwok, R. S., Chan, E. C., Hidalgo, S. and Chiu, J. C. (2023). CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters. PLoS Genet 19(2): e1010649. PubMed ID: 36809369
Summary:
Circadian clock and chromatin-remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. It has been previously reported that the BRAHMA (BRM) chromatin-remodeling complex promotes the repression of circadian gene expression in Drosophila. This study investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, rhythmic BRM binding to clock gene promoters was observed despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since it was previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), their effect on BRM occupancy to the period (per) promoter was examined. Reduced BRM binding to the DNA was observed in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, reduced BRM binding to the per promoter was observed in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. These conclusions are further supported by elevated BRM binding to the per promoter in flies subjected to constant light and experiments in Drosophila tissue culture in which the levels of CLK and TIM are manipulated. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin-remodeling complex.
Kyrchanova, O., Sokolov, V., Tikhonov, M., Schedl, P. and Georgiev, P. (2023). Transcriptional read through interrupts boundary function in Drosophila. bioRxiv. PubMed ID: 36824960
Summary:
In higher eukaryotes enhancer-promoter interactions are known to be restricted by the chromatin insulators/boundaries that delimit topologically associated domains (TADs); however, there are instances in which enhancer-promoter interactions span one or more boundary elements/TADs. At present, the mechanisms that enable cross-TAD regulatory interaction are not known. These studies hav taken advantage of the well characterized Drosophila Bithorax complex (BX-C) to study one potential mechanism for controlling boundary function and TAD organization. The regulatory domains of BX-C are flanked by boundaries which function to block crosstalk with their neighboring domains and also to support long distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, it was asked whether transcriptional readthrough can impact boundary function. For this purpose, advantage was taken of two BX-C boundary replacement platforms, Fab-7 (attP50) and F2 (attP) , in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. Boundary elements, promoters and polyadenylation signals arranged in different combinations were introduced and then assayed for boundary function. The results show that transcriptional readthrough can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is possible that many of them may function in the regulation of TAD organization.
Weinstein, M. L., Jaenke, C. M., Asma, H., Spangler, M., Kohnen, K. A., Konys, C. C., Williams, M. E., Williams, A. V., Rebeiz, M., Halfon, M. S. and Williams, T. M. (2023). A novel role for trithorax in the gene regulatory network for a rapidly evolving fruit fly pigmentation trait. PLoS Genet 19(2): e1010653. PubMed ID: 36795790
Summary:
Animal traits develop through the expression and action of numerous regulatory and realizator genes that comprise a gene regulatory network (GRN). For each GRN, its underlying patterns of gene expression are controlled by cis-regulatory elements (CREs) that bind activating and repressing transcription factors. These interactions drive cell-type and developmental stage-specific transcriptional activation or repression. Most GRNs remain incompletely mapped, and a major barrier to this daunting task is CRE identification. This study used an in silico method to identify predicted CREs (pCREs) that comprise the GRN which governs sex-specific pigmentation of Drosophila melanogaster. Through in vivo assays, it was demonstrated that many pCREs activate expression in the correct cell-type and developmental stage. Genome editing was employed to demonstrate that two CREs control the pupal abdomen expression of trithorax, whose function is required for the dimorphic phenotype. Surprisingly, trithorax had no detectable effect on this GRN's key trans-regulators, but shapes the sex-specific expression of two realizator genes. Comparison of sequences orthologous to these CREs supports an evolutionary scenario where these trithorax CREs predated the origin of the dimorphic trait. Collectively, this study demonstrates how in silico approaches can shed novel insights on the GRN basis for a trait's development and evolution.
Hamamoto, K., Umemura, Y., Makino, S. and Fukaya, T. (2023). Dynamic interplay between non-coding enhancer transcription and gene activity in development. Nat Commun 14(1): 826. PubMed ID: 36805453
Summary:
Non-coding transcription at the intergenic regulatory regions is a prevalent feature of metazoan genomes, but its biological function remains uncertain. This study devised a live-imaging system that permits simultaneous visualization of gene activity along with intergenic non-coding transcription at single-cell resolution in Drosophila. Quantitative image analysis reveals that elongation of RNA polymerase II across the internal core region of enhancers leads to suppression of transcriptional bursting from linked genes. Super-resolution imaging and genome-editing analysis further demonstrate that enhancer transcription antagonizes molecular crowding of transcription factors, thereby interrupting the formation of a transcription hub at the gene locus. This study also showed that a certain class of developmental enhancers is structurally optimized to co-activate gene transcription together with non-coding transcription effectively. It is suggested that enhancer function is flexibly tunable through the modulation of hub formation via surrounding non-coding transcription during development.
Xu, R., Dai, F., Wu, H., Jiao, R., He, F. and Ma, J. (2023). Shaping the scaling characteristics of gap gene expression patterns in Drosophila. Heliyon 9(2): e13623. PubMed ID: 36879745
Summary:
How patterns are formed to scale with tissue size remains an unresolved problem. This study investigated embryonic patterns of gap gene expression along the anterior-posterior (AP) axis in Drosophila. Embryos were used that greatly differed in length and, importantly, possess distinct length-scaling characteristics of the Bicoid (Bcd) gradient. The dynamic movements of gap gene expression boundaries were systematically analyze in relation to both embryo length and Bcd input as a function of time. The process through which such dynamic movements drive both an emergence of a global scaling landscape was shown, and evolution of boundary-specific scaling characteristics was documented. Despite initial differences in pattern scaling characteristics that mimic those of Bcd in the anterior, such characteristics of final patterns converge. This study thus partitions the contributions of Bcd input and regulatory dynamics inherent to the AP patterning network in shaping embryonic pattern's scaling characteristics.

Wednesday, August 23rd - RNA and Transposons

Nguyen, T. T. M., Munkhzul, C., Kim, J., Kyoung, Y., Vianney, M., Shin, S., Ju, S., Pham-Bui, H. A., Kim, J., Kim, J. S. and Lee, M. (2023). In vivo profiling of the Zucchini proximal proteome in the Drosophila ovary. Development 150(4). PubMed ID: 36762624
Summary:
PIWI-interacting RNAs (piRNAs) are small RNAs that play a conserved role in genome defense. The piRNA processing pathway is dependent on the sequestration of RNA precursors and protein factors in specific subcellular compartments. Therefore, a highly resolved spatial proteomics approach can help identify the local interactions and elucidate the unknown aspects of piRNA biogenesis. TurboID proximity labeling was performed to investigate the interactome of Zucchini (Zuc), a key factor of piRNA biogenesis in germline cells and somatic follicle cells of the Drosophila ovary. Quantitative mass spectrometry analysis of biotinylated proteins defined the Zuc-proximal proteome, including the well-known partners of Zuc. Many of these were enriched in the outer mitochondrial membrane (OMM), where Zuc was specifically localized. The proximal proteome of Zuc showed a distinct set of proteins compared with that of Tom20, a representative OMM protein, indicating that chaperone function-related and endomembrane system/vesicle transport proteins are previously unreported interacting partners of Zuc. The functional relevance of several candidates in piRNA biogenesis was validated by derepression of transposable elements after knockdown. These results present potential Zuc-interacting proteins, suggesting unrecognized biological processes.
Souidi, A., Nakamori, M., Zmojdzian, M., Jagla, T., Renaud, Y. and Jagla, K. (2023). Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1. EMBO Rep 24(4): e56616. PubMed ID: 36852954
Summary:
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. This study used Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. miR-1 down-regulation in the heart was found to lead to dilated cardiomyopathy (DCM), a DM1-associated phenotype. In silico screening for miR-1 targets was combined with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM.
Wang, Q., Trombley, S., Rashidzada, M. and Song, Y. (2023). Drosophila Laser Axotomy Injury Model to Investigate RNA Repair and Splicing in Axon Regeneration. Methods Mol Biol 2636: 401-419. PubMed ID: 36881313
Summary:
The limited axon regeneration capacity of mature neurons often leads to insufficient functional recovery after damage to the central nervous system (CNS). To promote CNS nerve repair, there is an urgent need to understand the regeneration machinery in order to develop effective clinical therapies. To this aim, this study developed a Drosophila sensory neuron injury model and the accompanying behavioral assay to examine axon regeneration competence and functional recovery after injury in the peripheral and central nervous systems. Specifically, a two-photon laser was used to induce axotomy and performed live imaging to assess axon regeneration, combined with the analysis of the thermonociceptive behavior as a readout of functional recovery. Using this model, it was found that the RNA 3'-terminal phosphate cyclase (Rtca), which acts as a regulator for RNA repair and splicing, responds to injury-induced cellular stress and impedes axon regeneration after axon breakage. How the Drosophila model is used to assess the role of Rtca during neuroregeneration is described.
Wang, L., Zhang, S., Hadjipanteli, S., Saiz, L., Nguyen, L., Silva, E. and Kelleher, E. (2023). P-element invasion fuels molecular adaptation in laboratory populations of Drosophila melanogaster. Evolution 77(4): 980-994. PubMed ID: 36749648
Summary:
Transposable elements (TEs) are mobile genetic parasites that frequently invade new host genomes through horizontal transfer. Invading TEs often exhibit a burst of transposition, followed by reduced transposition rates as repression evolves in the host. This study recreated the horizontal transfer of P-element DNA transposons into a Drosophila melanogaster host and followed the expansion of TE copies and evolution of host repression in replicate laboratory populations reared at different temperatures. It was observed that while populations maintained at high temperatures rapidly go extinct after TE invasion, those maintained at lower temperatures persist, allowing for TE spread and the evolution of host repression. It was also surprisingly discovered that invaded populations experienced recurrent insertion of P-elements into a specific long non-coding RNA, lncRNA:CR43651, and that these insertion alleles are segregating at unusually high frequency in experimental populations, indicative of positive selection. It is proposed that, in addition to driving the evolution of repression, transpositional bursts of invading TEs can drive molecular adaptation.
Silva, B., Picorelli, A. C. R. and Kuhn, G. C. S. (2023). In Silico Identification and Characterization of Satellite DNAs in 23 Drosophila Species from the Montium Group. Genes (Basel) 14(2). PubMed ID: 36833227
Summary:
Satellite DNA (satDNA) is a class of tandemly repeated non-protein coding DNA sequences which can be found in abundance in eukaryotic genomes. They can be functional, impact the genomic architecture in many ways, and their rapid evolution has consequences for species diversification. This study took advantage of the recent availability of sequenced genomes from 23 Drosophila species from the montium group to study their satDNA landscape. For this purpose, publicly available whole-genome sequencing Illumina reads and the TAREAN (tandem repeat analyzer) pipeline were used. The characterization of 101 non-homologous satDNA families in this group is reported, 93 of which are described for the first time. Their repeat units vary in size from 4 bp to 1897 bp, but most satDNAs show repeat units < 100 bp long and, among them, repeats ≤ 10 bp are the most frequent ones. The genomic contribution of the satDNAs ranges from ~1.4% to 21.6%. There is no significant correlation between satDNA content and genome sizes in the 23 species. At least one satDNA originated from an expansion of the central tandem repeats (CTRs) present inside a Helitron transposon. Finally, some satDNAs may be useful as taxonomic markers for the identification of species or subgroups within the group.
Orkenby, L., Skog, S., Ekman, H., Gozzo, A., Kugelberg, U., Ramesh, R., Magadi, S., Zambanini, G., Nordin, A., Cantu, C., Natt, D. and Ost, A. (2023). Stress-sensitive dynamics of miRNAs and Elba1 in Drosophila embryogenesis. Mol Syst Biol: e11148. PubMed ID: 36938679
Summary:
Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. This study shows that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, this study shows that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w(m4h) reporter. It is proposed that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.

Tuesday, August 22nd - Synapse and Vesicles

Marie, P. P., Fan, S. J., Mason, J., Wells, A., Mendes, C. C., Wainwright, S. M., Scott, S., Fischer, R., Harris, A. L., Wilson, C. and Goberdhan, D. C. I. (2023). Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation. J Extracell Vesicles 12(3): e12311. PubMed ID: 36872252
Summary:
Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7-positive multivesicular endosomes, and also in recycling Rab11a-positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos. Accessory ESCRT-III components have reported roles in ESCRT-III-mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT-III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a-enriched exosome preparations. These proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a-exosome production. Accessory ESCRT-III knockdown suppresses seminal fluid-mediated reproductive signalling by secondary cells and the growth-promoting activity of Rab11a-exosome-containing EVs from HCT116 cells. It is concluded that accessory ESCRT-III components have a specific, ubiquitin-independent role in Rab11a-exosome generation, a mechanism that might be targeted to selectively block pro-tumorigenic activities of these vesicles in cancer.
Sidisky, J. M., de Paula Moreira, D., Okumus, M., Caratenuto, R., Drost, C., Connors, B., Hussain, S., Alkhatib, S. and Babcock, D. T. (2023). Genome-wide analysis reveals novel regulators of synaptic maintenance in Drosophila. Genetics 223(4). PubMed ID: 36799927
Summary:
Maintaining synaptic communication is required to preserve nervous system function as an organism ages. While much work has been accomplished to understand synapse formation and development, Relatively little is understood regarding maintaining synaptic integrity throughout aging. To better understand the mechanisms responsible for maintaining synaptic structure and function, an unbiased forward genetic screen was performed to identify genes required for synapse maintenance of adult Drosophila neuromuscular junctions. Using flight behavior as a screening tool, this study evaluated flight ability in 198 lines from the Drosophila Genetic Reference Panel to identify single nucleotide polymorphisms (SNPs) that are associated with a progressive loss of flight ability with age. Among the many candidate genes identified from this screen, this study focussed on 10 genes with clear human homologs harboring SNPs that are most highly associated with synaptic maintenance. Functional validation of these genes using mutant alleles revealed a progressive loss of synaptic structural integrity. Tissue-specific knockdown of these genes using RNA interference (RNAi) uncovered important roles for these genes in either presynaptic motor neurons, postsynaptic muscles, or associated glial cells, highlighting the importance of each component of tripartite synapses. These results offer greater insight into the mechanisms responsible for maintaining structural and functional integrity of synapses with age.
Jun, Y. W., Lee, S., Ban, B. K., Lee, J. A. and Gao, F. B. (2023). Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis. Autophagy: 1-17. PubMed ID: 36849436
Summary:
Dysfunction of the endosomal sorting complex required for transport (ESCRT) has been linked to frontotemporal dementia (FTD) due in part to the accumulation of unsealed autophagosomes. However, the mechanisms of ESCRT-mediated membrane closure events on phagophores remain largely unknown. This study found that partial knockdown of non-muscle MYH10/myosin IIB/zip rescues neurodegeneration in both Drosophila and human iPSC-derived cortical neurons expressing FTD-associated mutant CHMP2B, a subunit of ESCRT-III. It was also found that MYH10 binds and recruits several autophagy receptor proteins during autophagosome formation induced by mutant CHMP2B or nutrient starvation. Moreover, MYH10 interacted with ESCRT-III to regulate phagophore closure by recruiting ESCRT-III to damaged mitochondria during PRKN/parkin-mediated mitophagy. Evidently, MYH10 is involved in the initiation of induced but not basal autophagy and also links ESCRT-III to mitophagosome sealing, revealing novel roles of MYH10 in the autophagy pathway and in ESCRT-related FTD pathogenesis.
Wang, L., Xu, Y., Yun, S., Yuan, Q., Satpute-Krishnan, P. and Ye, Y. (2023). SAYSD1 senses UFMylated ribosome to safeguard co-translational protein translocation at the endoplasmic reticulum.. Cell Rep 42(1): 112028. PubMed ID: 36848233
Summary:
Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating translocation-associated quality control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. This study conducted a genome-wide CRISPR-Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.
Perez-Moreno, J. J., Smith, R. C., Oliva, M. K., Gallo, F., Ojha, S., Muller, K. H. and O'Kane, C. J. (2023). Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics. J Cell Biol 222(6). PubMed ID: 36952540
Summary:
Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). It was therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. Reduced STIM-mediated ER-plasma membrane was found to contact underlie presynaptic Ca2+ defects in Rtnl1 mutants. These results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP.
Ghelani, T., Escher, M., Thomas, U., Esch, K., Lutzkendorf, J., Depner, H., Maglione, M., Parutto, P., Gratz, S., Matkovic-Rachid, T., Ryglewski, S., Walter, A. M., Holcman, D., O'Connor Giles, K., Heine, M. and Sigrist, S. J. (2023). Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca(2+) channels drives sustained active zone potentiation. Sci Adv 9(7): eade7804. PubMed ID: 36800417
Summary:
At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca(2+) channels (VGCCs). While AZs can potentiate SV release in the minutes range, an understanding of how AZ scaffold components and VGCCs engage into potentiation is lacking. This study establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. This single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Monday, August 21st - Embryonic Development

Riemondy, K., Henriksen, J. C. and Rissland, O. S. (2023). Intron dynamics reveal principles of gene regulation during the maternal-to-zygotic transition. Rna 29(5): 596-608. PubMed ID: 36764816
Summary:
The maternal-to-zygotic transition (MZT) is a conserved embryonic process in animals where developmental control shifts from the maternal to zygotic genome. A key step in this transition is zygotic transcription, and deciphering the MZT requires classifying newly transcribed genes. However, due to current technological limitations, this starting point remains a challenge for studying many species. This study presents an alternative approach that characterizes transcriptome changes based solely on RNA-seq data. By combining intron-mapping reads and transcript-level quantification, transcriptome dynamics were characterized during the Drosophila melanogaster MZT. This approach provides an accessible platform to investigate transcriptome dynamics that can be applied to the MZT in nonmodel organisms. In addition to classifying zygotically transcribed genes, this analysis revealed that over 300 genes express different maternal and zygotic transcript isoforms due to alternative splicing, polyadenylation, and promoter usage. The vast majority of these zygotic isoforms have the potential to be subject to different regulatory control, and over two-thirds encode different proteins. Thus, this analysis reveals an additional layer of regulation during the MZT, where new zygotic transcripts can generate additional proteome diversity.
Klonaros, D., Dresch, J. M. and Drewell, R. A. (2023). Transcriptome profile in Drosophila Kc and S2 embryonic cell lines. G3 (Bethesda). PubMed ID: 36869676
Summary:
Drosophila melanogaster cell lines are an important resource for a range of studies spanning genomics, molecular genetics and cell biology. Amongst these valuable lines are Kc167 and S2 cells, which were originally isolated in the late 1960s from embryonic sources and have been used extensively to investigate a broad spectrum of biological activities including cell-cell signaling and immune system function. Whole-genome tiling microarray analysis of total RNA from these two cell types was performed as part of the modENCODE project over a decade ago and revealed that they share a number of gene expression features. This study expands on these earlier studies by using deep coverage RNA sequencing approaches to investigate the transcriptional profile in Kc and S2 cells in detail. Comparison of the transcriptomes reveals that approximately 75% of the 13,919 annotated genes are expressed at a detectable level in at least one of the cell lines, with the majority of these genes expressed at high levels in both cell lines. Despite the overall similarity of the transcriptional landscape in the two cell types, 2588 differentially expressed genes are identified. Many of the genes with the largest fold change are known only by their "CG" designations, indicating that the molecular control of Kc and S2 cell identity may be regulated in part by a cohort of relatively uncharacterized genes. These data also indicate that both cell lines have distinct hemocyte-like identities, but share active signaling pathways and express a number of genes in the network responsible for dorsal-ventral patterning of the early embryo.
Villedieu, A., Alpar, L., Gaugue, I., Joudat, A., Graner, F., Bosveld, F. and Bellaiche, Y. (2023). Homeotic compartment curvature and tension control spatiotemporal folding dynamics. Nat Commun 14(1): 594. PubMed ID: 36737611
Summary:
Shape is a conspicuous and fundamental property of biological systems entailing the function of organs and tissues. While much emphasis has been put on how tissue tension and mechanical properties drive shape changes, whether and how a given tissue geometry influences subsequent morphogenesis remains poorly characterized. This study explored how curvature, a key descriptor of tissue geometry, impinges on the dynamics of epithelial tissue invagination. The morphogenesis of the fold separating the adult Drosophila head and thorax segments is driven by the invagination of the Deformed (Dfd) homeotic compartment. Dfd controls invagination by modulating actomyosin organization and in-plane epithelial tension via the Tollo and Dystroglycan receptors. By experimentally introducing curvature heterogeneity within the homeotic compartment, this study established that a curved tissue geometry converts the Dfd-dependent in-plane tension into an inward force driving folding. Accordingly, the interplay between in-plane tension and tissue curvature quantitatively explains the spatiotemporal folding dynamics. Collectively, this work highlights how genetic patterning and tissue geometry provide a simple design principle driving folding morphogenesis during development.
Nishizawa, K., Lin, S. Z., Chardes, C., Rupprecht, J. F. and Lenne, P. F. (2023). Two-point optical manipulation reveals mechanosensitive remodeling of cell-cell contacts in vivo. Proc Natl Acad Sci U S A 120(13): e2212389120. PubMed ID: 36947511
Summary:
Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding understanding of how mechanical stresses remodel cell-cell contacts. This study developed a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows production of junction extension and shrinkage through different push and pull manipulations at the edges of junctions. These observations were used to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, this study showed that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether this work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling.
Perez-Mojica, J. E., Enders, L., Walsh, J., Lau, K. H. and Lempradl, A. (2023). Continuous transcriptome analysis reveals novel patterns of early gene expression in Drosophila embryos. Cell Genom 3(3): 100265. PubMed ID: 36950383
Summary:
The transformative events during early organismal development lay the foundation for body formation and long-term phenotype. The rapid progression of events and the limited material available present major barriers to studying these earliest stages of development. This study reports an operationally simple RNA sequencing approach for high-resolution, time-sensitive transcriptome analysis in early (≤3 h) Drosophila embryos. This method does not require embryo staging but relies on single-embryo RNA sequencing and transcriptome ordering along a developmental trajectory (pseudo-time). The resulting high-resolution, time-sensitive mRNA expression profiles reveal the exact onset of transcription and degradation for thousands of transcripts. Further, using sex-specific transcription signatures, embryos can be sexed directly, eliminating the need for Y chromosome genotyping and revealing patterns of sex-biased transcription from the beginning of zygotic transcription. These data provide an unparalleled resolution of gene expression during early development and enhance the current understanding of early transcriptional processes.
Xue, Y., Krishnan, A., Chahda, J. S., Schweickart, R. A., Sousa-Neves, R. and Mizutani, C. M. (2023). The epithelial polarity genes frazzled and GUK-holder adjust morphogen gradients to coordinate changes in cell position with cell fate specification. PLoS Biol 21(3): e3002021. PubMed ID: 36913435
Summary:
Morphogenetic gradients specify distinct cell populations within tissues. Originally, morphogens were conceived as substances that act on a static field of cells, yet cells usually move during development. Thus, the way cell fates are defined in moving cells remains a significant and largely unsolved problem. This issue was investigated using spatial referencing of cells and 3D spatial statistics in the Drosophila blastoderm to reveal how cell density responds to morphogenetic activity. The morphogen Decapentaplegic (DPP) attracts cells towards its peak levels in the dorsal midline, whereas Dorsal (DL) stalls them ventrally. Frazzled and GUK-holder were identified as the downstream effectors regulated by these morphogens that constrict cells and provide the mechanical force necessary to draw cells dorsally. Surprisingly, GUKH and FRA modulate the DL and DPP gradient levels and this regulation creates a very precise mechanism of coordinating cell movement and fate specification.

Friday, August 18th - Behavior

Guo, L., Sun, Y. and Liu, S. (2023). Adaptive behaviors of Drosophila larvae on slippery surfaces. J Biol Phys 49(1): 121-132. PubMed ID: 36790728
Summary:
Friction is ubiquitous but an essential force for insects during locomotion. Insects use dedicated bio-mechanical systems such as adhesive pads to modulate the intensity of friction, providing a stable grip with touching substrates for locomotion. However, how to uncover behavioral adaptation and regulatory neural circuits of friction modification is still largely understood. A novel behavior paradigm was devised in this study to investigate adaptive behavioral alternation of Drosophila larvae under low-friction surfaces. A tail looseness phenotype similar to slipping behavior in humans, was found to be a primary indicator to assess the degree of slipping. A gradual reduction on slipping level was found in wild-type larvae after successive larval crawling, coupled with incremental tail contraction, displacement, and speed acceleration. Meanwhile, a strong correlation was found between tail looseness index and length of contraction, suggesting that lengthening tail contraction may contribute to enlarging the contact area with the tube. Moreover, a delayed adaptation was found in rut mutant larvae, inferring that neural plasticity may participate in slipping adaptation. In conclusion, this paradigm can be easily and reliably replicated, providing a feasible pathway to uncover the behavioral principle and neural mechanism of acclimation of Drosophila larvae to low-friction conditions.
Tolassy, V., Cazale-Debat, L., Houot, B., Reynaud, R., Heydel, J. M., Ferveur, J. F. and Everaerts, C. (2023). Drosophila Free-Flight Odor Tracking is Altered in a Sex-Specific Manner By Preimaginal Sensory Exposure. J Chem Ecol 49(3-4): 179-194. PubMed ID: 36881326
Summary:
In insects such as Drosophila melanogaster, flight guidance is based on converging sensory information provided by several modalities, including chemoperception. Drosophila flies are particularly attracted by complex odors constituting volatile molecules from yeast, pheromones and microbe-metabolized food. This study asked whether early preimaginal exposure to maternally transmitted egg factors could affect free-flight odor tracking in flies of both sexes. The main experiment consisted of testing flies differently conditioned during preimaginal development in a wind tunnel. Each fly was presented with a dual choice of food labeled by groups of each sex of D. melanogaster or D. simulans flies. The combined effect of food with the cis-vaccenyl acetate pheromone (cVA), which is involved in aggregation behavior, was also measured. Moreover, the headspace method was used to determine the "odorant" identity of the different labeled foods tested. The antennal electrophysiological response to cVA in females and males resulting from the different preimaginal conditioning procedures was also measured. The data indicate that flies differentially modulated their flight response (take off, flight duration, food landing and preference) according to sex, conditioning and food choice. Headspace analysis revealed that many food-derived volatile molecules diverged between sexes and species. Antennal responses to cVA showed clear sex-specific variation for conditioned flies but not for control flies. In summary, this study indicates that preimaginal conditioning can affect Drosophila free flight behavior in a sex-specific manner.
Sun, J., Liu, W. K., Ellsworth, C., Sun, Q., Pan, Y. F., Huang, Y. C. and Deng, W. M. (2023). Integrating lipid metabolism, pheromone production and perception by Fruitless and Hepatocyte nuclear factor 4. bioRxiv. PubMed ID: 36865119
Summary:
Sexual attraction and perception, governed by separate genetic circuits in different organs, are crucial for mating and reproductive success, yet the mechanisms of how these two aspects are integrated remain unclear. In Drosophila, the male-specific isoform of Fruitless (Fru), Fru (M), is known as a master neuro-regulator of innate courtship behavior to control perception of sex pheromones in sensory neurons. This study shows that the non-sex specific Fru isoform (Fru (COM)) is necessary for pheromone biosynthesis in hepatocyte-like oenocytes for sexual attraction. Loss of Fru (COM) in oenocytes resulted in adults with reduced levels of the cuticular hydrocarbons (CHCs), including sex pheromones; adults showed altered sexual attraction and reduced cuticular hydrophobicity. Hepatocyte nuclear factor 4 (Hnf4) was identified as a key target of Fru (COM) in directing fatty acid conversion to hydrocarbons in adult oenocytes. fru- and Hnf4 -depletion disrupts lipid homeostasis, resulting in a novel sex-dimorphic CHC profile, which differs from doublesex - and transformer -dependent sexual dimorphism of the CHC profile. Thus, Fru couples pheromone perception and production in separate organs for precise coordination of chemosensory communication that ensures efficient mating behavior.
Odell, S. R., Zito, N., Clark, D. and Mathew, D. (2023). Stability of olfactory behavior syndromes in the Drosophila larva. Sci Rep 13(1): 2398. PubMed ID: 36765192
Summary:
Individuals of many animal populations exhibit idiosyncratic behaviors. One measure of idiosyncratic behavior is a behavior syndrome, defined as the stability of one or more behavior traits in an individual across different situations. While behavior syndromes have been described in various animal systems, their properties and the circuit mechanisms that generate them are poorly understood. Thus there is an incomplete understanding of how circuit properties influence animal behavior. This study characterize olfactory behavior syndromes in the Drosophila larva. Larvae were shown to exhibit idiosyncrasies in their olfactory behavior over short time scales. They are influenced by the larva's satiety state and odor environment. Additionally, a group of antennal lobe local neurons was identified that influence the larva's idiosyncratic behavior. These findings reveal previously unsuspected influences on idiosyncratic behavior. They further affirm the idea that idiosyncrasies are not simply statistical phenomena but manifestations of neural mechanisms. In light of these findings, the importance of idiosyncrasies to animal survival and how they might be studied was discussed more broadly.
Cooney, P. C., Li, W., Huang, Y., Hormigo, R., Tabachnik, T., Hillman, E. M. C., Grueber, W. B. and Zarin, A. A. (2023). Neuromuscular Basis of Drosophila Larval Escape Behavior. bioRxiv. PubMed ID: 36778508
Summary:
When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behavior. In Drosophila larvae, escape behavior is characterized by C-shaped bending and lateral rolling, followed by rapid forward crawling. The sensory circuitry that promotes escape has been extensively characterized, but the motor programs underlying escape are unknown. This study characterized the neuromuscular basis of escape. High-speed, volumetric, Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy was used to image muscle activity during larval rolling. Unlike the sequential peristaltic muscle contractions from segment to segment that underlie forward and backward crawling, muscle activity progresses in a circumferential sequence during bending and rolling. Certain muscle subgroups show functional antagonism during bending and rolling. EM connectome data was used to identify premotor to motor connectivity patterns that could drive rolling behavior, and the necessity of specific groups of motor neurons in rolling using neural silencing approaches was tested. The data reveal the body-wide muscle activity patterns and putative premotor circuit organization for escape.
Eichler, K., Hampel, S., Alejandro-Garcia, A., Calle-Schuler, S. A., Santana-Cruz, A., Kmecova, L., Blagburn, J. M., Hoopfer, E. D. and Seeds, A. M. (2023). Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila. bioRxiv. PubMed ID: 36798384
Summary:
Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. Previous work has shown that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust. This study identified nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming.

Thusday, August 17th - Gonads

Talbot, D. E., Vormezeele, B. J., Kimble, G. C., Wineland, D. M., Kelpsch, D. J., Giedt, M. S. and Tootle, T. L. (2023). Prostaglandins limit nuclear actin to control nucleolar function during oogenesis. Front Cell Dev Biol 11: 1072456. PubMed ID: 36875757
Summary:
Prostaglandins (PGs), locally acting lipid signals, regulate female reproduction, including oocyte development. However, the cellular mechanisms of PG action remain largely unknown. One cellular target of PG signaling is the nucleolus. Indeed, across organisms, loss of PGs results in misshapen nucleoli, and changes in nucleolar morphology are indicative of altered nucleolar function. A key role of the nucleolus is to transcribe ribosomal RNA (rRNA) to drive ribosomal biogenesis. This study takes advantage of the robust, in vivo system of Drosophila oogenesis to define the roles and downstream mechanisms whereby PGs regulate the nucleolus. The altered nucleolar morphology due to PG loss is not due to reduced rRNA transcription. Instead, loss of PGs results in increased rRNA transcription and overall protein translation. PGs modulate these nucleolar functions by tightly regulating nuclear actin, which is enriched in the nucleolus. Specifically, this study found that loss of PGs results in both increased nucleolar actin and changes in its form. Increasing nuclear actin, by either genetic loss of PG signaling or overexpression of nuclear targeted actin (NLS-actin), results in a round nucleolar morphology. Further, loss of PGs, overexpression of NLS-actin or loss of Exportin 6, all manipulations that increase nuclear actin levels, results in increased RNAPI-dependent transcription. Together these data reveal PGs carefully balance the level and forms of nuclear actin to control the level of nucleolar activity required for producing fertilization competent oocytes.
Hafezi, Y., Omurzakov, A., Carlisle, J. A., Caldas, I. V., Wolfner, M. F. and Clark, A. G. (2023). The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract. bioRxiv. PubMed ID: 36778485
Summary:
Unique patterns of inheritance and selection on Y chromosomes lead to the evolution of specialized gene functions. Yet characterizing the function of genes on Y chromosomes is notoriously difficult. This study reports CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. WDY mutants produce mature sperm with beating tails that can be transferred to females but fail to enter the female sperm storage organs. The sperm tails of WDY mutants beat approximately half as fast as wild-type sperm's and that the mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract (RT). These specific motility defects likely cause the sperm storage defect and sterility of the mutants. Regional and genotype-dependent differences in sperm motility suggest that sperm tail beating and propulsion do not always correlate. Furthermore, significant differences were observed in the hydrophobicity of key residues of a putative calcium-binding domain between orthologs of WDY that are Y-linked and those that are autosomal. Given that WDY appears to be evolving under positive selection, these results suggest that WDY 's functional evolution coincides with its transition from autosomal to Y-linked in Drosophila melanogaster and its most closely related species. Finally, it was shown that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. In contrast to WDY, PRY mutants do swim in the female RT, suggesting they are defective in yet another mode of motility, navigation, or a necessary interaction with the female RT. Overall, this study provides direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.
Parsons, T. T., Mosallaei, S. and Raftery, L. A. (2023). Two phases for centripetal migration of Drosophila melanogaster follicle cells: initial ingression followed by epithelial migration.. Development 150(6). PubMed ID: 36807509
Summary:
During Drosophila oogenesis, somatic follicle cells (FCs) differentiate to secrete components of the eggshell. Before secretion, the epithelium reorganizes to shape eggshell specializations, including border FC collective cell migration and later dorsal formation. These FC movements provide valuable insights into collective cell migration. However, little is known about centripetal migration, which encloses the oocyte after secretion has begun. Centripetal migration begins with apical extension of a few FCs that move away from the basement membrane to invade between germ cells. This study defines a timeline of reproducible milestones, using time-lapse imaging of egg chamber explants. Inward migration occurs in two phases. First, leading centripetal FCs ingress, extending apically over the anterior oocyte, and constricting basally. Second, following FCs move collectively toward the anterior, then around the corner to move inward with minimal change in aspect ratio. E-cadherin was required in leading centripetal FCs for their normal ingression, assessed with homozygous shotgun mutant or RNAi knockdown clones; ingression was influenced non-autonomously by mutant following FCs. This work establishes centripetal migration as an accessible model for biphasic E-cadherin-adhesion-mediated collective migration.
Saha, B., Acharjee, S., Ghosh, G., Dasgupta, P. and Prasad, M. (2023). Germline protein, Cup, non-cell autonomously limits migratory cell fate in Drosophila oogenesis. PLoS Genet 19(2): e1010631. PubMed ID: 36791149
Summary:
Specification of migratory cell fate from a stationary population is complex and indispensable both for metazoan development as well for the progression of the pathological condition like tumor metastasis. Though this cell fate transformation is widely prevalent, the molecular understanding of this phenomenon remains largely elusive. This study employed the model of border cells (BC) in Drosophila oogenesis and identified germline activity of an RNA binding protein, Cup that limits acquisition of migratory cell fate from the neighbouring follicle epithelial cells. As activation of JAK-STAT in the follicle cells is critical for BC specification, these data suggest that Cup, non-cell autonomously restricts the domain of JAK-STAT by activating Notch in the follicle cells. Employing genetics and Delta endocytosis assay, Cup was demonstrated to regulate Delta recycling in the nurse cells through Rab11GTPase thus facilitating Notch activation in the adjacent follicle cells. Since Notch and JAK-STAT are antagonistic, it is proposed that germline Cup functions through Notch and JAK-STAT to modulate BC fate specification from their static epithelial progenitors.
Price, K. L., Tharakan, D. M. and Cooley, L. (2023). Evolutionarily conserved midbody remodeling precedes ring canal formation during gametogenesis. Dev Cell 58(6): 474-488.e475. PubMed ID: 36898376
Summary:
How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges, called "ring canals," is poorly understood. Using time-lapse imaging in Drosophila, this study observe that ring canal formation occurs through extensive remodeling of the germ cell midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germ cell midbody cores reorganize and join the midbody ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function to stabilize the midbody, similar to its role during somatic cell cytokinesis. These results provide important insights into the broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.
Hoshino, R., Sano, H., Yoshinari, Y., Nishimura, T. and Niwa, R. (2023). Circulating fructose regulates a germline stem cell increase via gustatory receptor-mediated gut hormone secretion in mated Drosophila. Sci Adv 9(8): eadd5551. PubMed ID: 36827377
Summary:
<Oogenesis is influenced by multiple environmental factors. In the fruit fly, Drosophila melanogaster, nutrition and mating have large impacts on an increase in female germline stem cells (GSCs). However, it is unclear whether these two factors affect this GSC increase interdependently. This study reports that dietary sugars are crucial for the GSC increase after mating. Dietary glucose is required for mating-induced release of neuropeptide F (NPF) from enteroendocrine cells (EECs), followed by NPF-mediated enhancement of GSC niche signaling. Unexpectedly, dietary glucose does not directly act on NPF-positive EECs. Rather, it contributes to elevation of hemolymph fructose generated through the polyol pathway. Elevated fructose stimulates the fructose-specific gustatory receptor, Gr43a, in NPF-positive EECs, leading to NPF secretion. This study demonstrates that circulating fructose, derived from dietary sugars, is a prerequisite for the GSC increase that leads to enhancement of egg production after mating.

Wednesday, August 16th - Disease Models

Rimal, S., Tantray, I., Li, Y., Pal Khaket, T., Li, Y., Bhurtel, S., Li, W., Zeng, C. and Lu, B. (2023). Reverse electron transfer is activated during aging and contributes to aging and age-related disease. EMBO Rep 24(4): e55548. PubMed ID: 36794623
Summary:
Mechanisms underlying the depletion of NAD(+) and accumulation of reactive oxygen species (ROS) in aging and age-related disorders remain poorly defined. This study shows that reverse electron transfer (RET) at mitochondrial complex I, which causes increased ROS production and NAD(+) to NADH conversion and thus lowered NAD(+) /NADH ratio, is active during aging. Genetic or pharmacological inhibition of RET decreases ROS production and increases NAD(+) /NADH ratio, extending the lifespan of normal flies. The lifespan-extending effect of RET inhibition is dependent on NAD(+) -dependent Sirtuin, highlighting the importance of NAD(+) /NADH rebalance, and on longevity-associated Foxo and autophagy pathways. RET and RET-induced ROS and NAD(+) /NADH ratio changes are prominent in human induced pluripotent stem cell (iPSC) model and fly models of Alzheimer's disease (AD). Genetic or pharmacological inhibition of RET prevents the accumulation of faulty translation products resulting from inadequate ribosome-mediated quality control, rescues relevant disease phenotypes, and extends the lifespan of Drosophila and mouse AD models. Deregulated RET is therefore a conserved feature of aging, and inhibition of RET may open new therapeutic opportunities in the context of aging and age-related diseases including AD.
Rodrigues, A., MacQuarrie, K. L., Freeman, E., Lin, A., Willis, A. B., Xu, Z., Alvarez, A. A., Ma, Y., White, B. E. P., Foltz, D. R. and Huang, S. (2023). Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer. Mol Biol Cell 34(4): br5. PubMed ID: 36753381
Summary:
Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, this study examined nucleolar structure and centromeres at various differentiation stages using cell culture models, and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.
Rasheed, M. Z., Khatoon, R., Talat, F., Alam, M. M., Tabassum, H. and Parvez, S. (2023). Melatonin Mitigates Rotenone-Induced Oxidative Stress and Mitochondrial Dysfunction in the Drosophila melanogaster Model of Parkinson's Disease-like Symptoms. ACS Omega 8(8): 7279-7288. PubMed ID: 36872990
Summary:
Parkinson's disease (PD) is the second most common neurodegenerative disorder; however, its etiology remains elusive. Antioxidants are considered to be a promising approach for decelerating neurodegenerative disease progression owing to extensive examination of the relationship between oxidative stress and neurodegenerative diseases. This study investigated the therapeutic effect of melatonin against rotenone-induced toxicity in the Drosophila model of PD. The 3-5 day old flies were divided into four groups: control, melatonin alone, melatonin and rotenone, and rotenone alone groups. According to their respective groups, flies were exposed to a diet containing rotenone and melatonin for 7 days. Melatonin was found to significantly reduced the mortality and climbing ability of Drosophila because of its antioxidative potency. It alleviated the expression of Bcl 2, tyrosine hydroxylase (TH), NADH dehydrogenase, mitochondrial membrane potential, and mitochondrial bioenergetics and decreased caspase 3 expression in the Drosophila model of rotenone-induced PD-like symptoms. These results indicate the neuromodulatory effect of melatonin, and that it is likely modulated against rotenone-induced neurotoxicity by suppressing oxidative stress and mitochondrial dysfunctions.
Ren, J., Zeng, Q., Wu, H., Liu, X., Guida, M. C., Huang, W., Zhai, Y., Li, J., Ocorr, K., Bodmer, R. and Tang, M. (2023). Deacetylase-dependent and -independent role of HDAC3 in cardiomyopathy. J Cell Physiol 238(3): 647-658. PubMed ID: 36745702
Summary:
Cardiomyopathy is a common disease of cardiac muscle that negatively affects cardiac function. HDAC3 commonly functions as corepressor by removing acetyl moieties from histone tails. However, a deacetylase-independent role of HDAC3 has also been described. Cardiac deletion of HDAC3 causes reduced cardiac contractility accompanied by lipid accumulation, but the molecular function of HDAC3 in cardiomyopathy remains unknown. This study has used powerful genetic tools in Drosophila to investigate the enzymatic and nonenzymatic roles of HDAC3 in cardiomyopathy. Using the Drosophila heart model, it was shown that cardiac-specific HDAC3 knockdown (KD) leads to prolonged systoles and reduced cardiac contractility. Immunohistochemistry revealed structural abnormalities characterized by myofiber disruption in HDAC3 KD hearts. Cardiac-specific HDAC3 KD showed increased levels of whole-body triglycerides and increased fibrosis. The introduction of deacetylase-dead HDAC3 mutant in HDAC3 KD background showed comparable results with wild-type HDAC3 in aspects of contractility and Pericardin deposition. However, deacetylase-dead HDAC3 mutants failed to improve triglyceride accumulation. These data indicate that HDAC3 plays a deacetylase-independent role in maintaining cardiac contractility and preventing Pericardin deposition as well as a deacetylase-dependent role to maintain triglyceride homeostasis.
Sheng, L., Shields, E. J., Gospocic, J., Sorida, M., Ju, L., Byrns, C. N., Carranza, F., Berger, S. L., Bonini, N. and Bonasio, R. (2023). Ensheathing glia promote increased lifespan and healthy brain aging. Aging Cell: e13803. PubMed ID: 36840361
Summary:
Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian microglia. Changes in glia composition over the life of ants and fruit flies have been reported, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. This study shows that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid-beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity.
Sousa, A., Rocha, S., Vieira, J., Reboiro-Jato, M., Lopez-Fernandez, H. and Vieira, C. P. (2023). On the identification of potential novel therapeutic targets for ataxia type 1 (SCA1) neurodegenerative disease using EvoPPI3. J Integr Bioinform. PubMed ID: 36848492
Summary:
EvoPPI, a meta-database for protein-protein interactions (PPI), has been upgraded (EvoPPI3) to accept new types of data, namely, PPI from patients, cell lines, and animal models, as well as data from gene modifier experiments, for nine neurodegenerative polyglutamine (polyQ) diseases caused by an abnormal expansion of the polyQ tract. The integration of the different types of data allows users to easily compare them, as shown for Ataxin-1, the polyQ protein involved in spinocerebellar ataxia type 1 (SCA1) disease. Using all available datasets and the data obtained for Drosophila melanogaster wt and exp Ataxin-1 mutants (also available at EvoPPI3), this study showed that, in humans, the Ataxin-1 network is much larger than previously thought (380 interactors), with at least 909 interactors. The functional profiling of the newly identified interactors is similar to the ones already reported in the main PPI databases. 16 out of 909 interactors are putative novel SCA1 therapeutic targets, and all but one are already being studied in the context of this disease. The 16 proteins are mainly involved in binding and catalytic activity (mainly kinase activity), functional features already thought to be important in the SCA1 disease.

Tuesday, August 15th - Tumors, Cancer and Growth

Seong, C. S., Huang, C., Boese, A. C., Hou, Y., Koo, J., Mouw, J. K., Rupji, M., Joseph, G., Johnston, H. R., Claussen, H., Switchenko, J. M., Behera, M., Churchman, M., Kolesar, J. M., Arnold, S. M., Kerrigan, K., Akerley, W., Colman, H., Johns, M. A., Arciero, C., Zhou, W., Marcus, A. I., Ramalingam, S. S., Fu, H. and Gilbert-Ross, M. (2023). Loss of the endocytic tumor suppressor HD-PTP phenocopies LKB1 and promotes RAS-driven oncogenesis. bioRxiv. PubMed ID: 36747658
Summary:
Oncogenic RAS mutations drive aggressive cancers that are difficult to treat in the clinic, and while direct inhibition of the most common KRAS variant in lung adenocarcinoma (G12C) is undergoing clinical evaluation, a wide spectrum of oncogenic RAS variants together make up a large percentage of untargetable lung and GI cancers. This study reports that loss-of-function alterations (mutations and deep deletions) in the gene that encodes HD-PTP (PTPN23) occur in up to 14% of lung cancers in the ORIEN Avatar lung cancer cohort, associate with adenosquamous histology, and occur alongside an altered spectrum of KRAS alleles. Furthermore, this study shows that in publicly available early-stage NSCLC studies loss of HD-PTP is mutually exclusive with loss of LKB1, which suggests they restrict a common oncogenic pathway in early lung tumorigenesis. In support of this, knockdown of HD-PTP in RAS-transformed lung cancer cells is sufficient to promote FAK-dependent invasion. Lastly, knockdown of the Drosophila homolog of HD-PTP (dHD-PTP/Myopic) synergizes to promote RAS-dependent neoplastic progression. These findings highlight a novel tumor suppressor that can restrict RAS-driven lung cancer oncogenesis and identify a targetable pathway for personalized therapeutic approaches for adenosquamous lung cancer.
Datta, I., Vassel, T., Linkous, B., Odum, T., Drew, C., Taylor, A. and Bangi, E. (2023). A targeted genetic modifier screen in Drosophila uncovers vulnerabilities in a genetically complex model of colon cancer. G3 (Bethesda). PubMed ID: 36880303
Summary:
Kinases are key regulators of cellular signal transduction pathways. Many diseases including cancer, are associated with global alterations in protein phosphorylation networks. As a result, kinases are frequent targets of drug discovery efforts. However, target identification and assessment, a critical step in targeted drug discovery that involves identifying essential genetic mediators of disease phenotypes, can be challenging in complex, heterogeneous diseases like cancer where multiple concurrent genomic alterations are common. Drosophila is a particularly useful genetic model system to identify novel regulators of biological processes through unbiased genetic screens. This study reports two classic genetic modifier screens focusing on the Drosophila kinome to identify kinase regulators in two different backgrounds: KRAS TP53 PTEN APC, a multigenic cancer model that targets four genes recurrently mutated in human colon tumors and KRAS alone, a simpler model that targets one of the most frequently altered pathways in cancer. These screens identified hits unique to each model and one shared by both, emphasizing the importance of capturing the genetic complexity of human tumor genome landscapes in experimental models. Follow-up analysis of two hits from the KRAS-only screen suggests that classical genetic modifier screens in heterozygous mutant backgrounds that result in a modest, non-lethal reduction in candidate gene activity in the context of a whole animal - a key goal of systemic drug treatment - may be a particularly useful approach to identify most rate-limiting genetic vulnerabilities in disease models as ideal candidate drug targets.
Faria, L., Canato, S., Jesus, T. T., Goncalves, M., Guerreiro, P. S., Lopes, C. S., Meireles, I., Morais-de-Sa, E., Paredes, J. and Janody, F. (2023). Activation of an actin signaling pathway in pre-malignant mammary epithelial cells by P-cadherin is essential for transformation. Dis Model Mech 16(2). PubMed ID: 36808468
Summary:
Alterations in the expression or function of cell adhesion molecules have been implicated in all steps of tumor progression. Among those, P-cadherin is highly enriched in basal-like breast carcinomas, playing a central role in cancer cell self-renewal, collective cell migration and invasion. To establish a clinically relevant platform for functional exploration of P-cadherin effectors in vivo, a humanized P-cadherin Drosophila model was generated. This study reports that actin nucleators, Mrtf and Srf, are main P-cadherin effectors in the fly. These findings were validated in a human mammary epithelial cell line with conditional activation of the SRC oncogene. Prior to promoting malignant phenotypes, SRC induces a transient increase in P-cadherin expression, which correlates with MRTF-A accumulation, its nuclear translocation and the upregulation of SRF target genes. Moreover, knocking down P-cadherin, or preventing F-actin polymerization, impairs SRF transcriptional activity. Furthermore, blocking MRTF-A nuclear translocation hampers proliferation, self-renewal and invasion. Thus, in addition to sustaining malignant phenotypes, P-cadherin can also play a major role in the early stages of breast carcinogenesis by promoting a transient boost of MRTF-A-SRF signaling through actin regulation.
La Marca, J. E., Ely, R. W., Diepstraten, S. T., Burke, P., Kelly, G. L., Humbert, P. O. and Richardson, H. E. (2023). A Drosophila chemical screen reveals synergistic effect of MEK and DGKα inhibition in Ras-driven cancer. Dis Model Mech 16(3). PubMed ID: 36861754
Summary:
Elevated Ras signalling is highly prevalent in human cancer; however, targeting Ras-driven cancers with Ras pathway inhibitors often leads to undesirable side effects and to drug resistance. Thus, identifying compounds that synergise with Ras pathway inhibitors would enable lower doses of the Ras pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a specialised chemical screen using a Drosophila model of Ras-driven cancer, this study has identified compounds that reduce tumour size by synergising with sub-therapeutic doses of the Ras pathway inhibitor trametinib, which targets MEK, the mitogen-activated protein kinase kinase, in this pathway. Analysis of one of the hits, ritanserin, and related compounds revealed that diacyl glycerol kinase α (DGKα, Dgk in Drosophila) was the critical target required for synergism with trametinib. Human epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene SCRIB were also sensitive to treatment with trametinib and DGKα inhibitors. Mechanistically, DGKα inhibition synergises with trametinib by increasing the P38 stress-response signalling pathway in H-RASG12V SCRIBRNAi cells, which could lead to cell quiescence. These results reveal that targeting Ras-driven human cancers with Ras pathway and DGKα inhibitors should be an effective combination drug therapy.
Cabrera, A. J. H., Gumbiner, B. M. and Kwon, Y. V. (2023). Remodeling of E-cadherin subcellular localization during cell dissemination. Mol Biol Cell 34(5): ar46. PubMed ID: 36989029
Summary:
Given the role of E-cadherin (E-cad) in holding epithelial cells together, an inverse relationship between E-cad levels and cell invasion during the epithelial-mesenchymal transition and cancer metastasis has been well recognized. This study reports that E-cad is necessary for the invasiveness of Ras(V12)-transformed intestinal epithelial cells in Drosophila. E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions--the actin- and cortactin-rich invadopodium-like protrusions associated with the breach of the extracellular matrix (ECM)--during dissemination of Ras(V12)-transformed intestinal epithelial cells. Loss of E-cad impairs the elongation of invasive protrusions and attenuates the ability of Ras(V12)-transformed cells to compromise the ECM. Notably, E-cad and cortactin affect each other's localization to invasive protrusions. Given the essential roles of cortactin in cell invasion, these observations indicate that E-cad plays a role in the invasiveness of Ras(V12)-transformed intestinal epithelial cells by controlling cortactin localization to invasive protrusions. Thus this study demonstrates that E-cad is a component of invasive protrusions and provides molecular insights into the unconventional role of E-cad in cell dissemination in vivo.
Song, F., Zhang, W., Li, X., Chen, X., Yuan, X., Jiang, M., Zhao, Y., Liu, Q. and Zhou, Z. (2023). FSBP suppresses tumor cell migration by inhibiting the JNK pathway. iScience 26(4): 106440. PubMed ID: 37035004
Summary:
The main cause of high mortality in cancer patients is tumor metastasis. Exploring the underlying mechanism of tumor metastasis is of great significance for clinical treatments. This study has identified the transcription factor Apt/FSBP is a suppressor for tumor metastasis. In Drosophila wing disc, knockdown of apt is able to trigger cell migration, whereas overexpression of apt hampers scrib-RNAi-induced tumor cell migration. Further studies show that loss of apt promotes cell migration JNK pathway . To investigate the role of FSBP, the homolog of Apt in mammals, Fsbp liver-specific knockout mice were constructed. Knockout of Fsbp in liver does not cause any detectable physiological defects, but predisposes to tumorigenesis on DEN and CCl(4) treatment. In addition, loss of Fsbp accelerates tumor metastasis from liver to diaphragm. Taken together, this study uncovers FSBP is a novel tumor suppressor, and provides it as a considerable drug target for tumor treatment.

Monday, August 14th - Signaling

Qian, W., Guo, M., Peng, J., Zhao, T., Li, Z., Yang, Y., Li, H., Zhang, X., King-Jones, K. and Cheng, D. (2023). Decapentaplegic retards lipolysis during metamorphosis in Bombyx mori and Drosophila melanogaster. Insect Biochem Mol Biol 155: 103928. PubMed ID: 36870515
Summary:
Insect morphogen Decapentaplegic (Dpp) functions as one of the key extracellular ligands of the Bone Morphogenetic Protein (BMP) signaling pathway. Previous studies in insects mainly focused on the roles of Dpp during embryonic development and the formation of adult wings. This study demonstrated a new role for Dpp in retarding lipolysis during metamorphosis in both Bombyx mori and Drosophila melanogaster. CRISPR/Cas9-mediated mutation of Bombyx dpp causes pupal lethality, induces an excessive and premature breakdown of lipids in the fat body, and upregulates the expressions of several lipolytic enzyme genes, including brummer (bmm), lipase 3 (lip3), and hormone-sensitive lipase (hsl), and lipid storage droplet 1 (lsd1), a lipid droplets (LD)-associated protein gene. Further investigation in Drosophila reveals that salivary gland-specific knockdown of the dpp gene and fat body-specific knockdown of Mad involved in Dpp signaling phenocopy the effects of Bombyx dpp mutation on pupal development and lipolysis. Taken together, these data indicate that the Dpp-mediated BMP signaling in the fat body maintains lipid homeostasis by retarding lipolysis, which is necessary for pupa-adult transition during insect metamorphosis.
Scholz, N., Dahse, A. K., Kemkemer, M., Bormann, A., Auger, G. M., Vieira Contreras, F., Ernst, L. F., Staake, H., Korner, M. B., Buhlan, M., Meyer-Molck, A., Chung, Y. K., Blanco-Redondo, B., Klose, F., Jarboui, M. A., Ljaschenko, D., Bigl, M. and Langenhan, T. (2023). Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation. Nature 615(7954): 945-953. PubMed ID: 36890234
Summary:
Adhesion G-protein-coupled receptors (aGPCRs) bear notable similarity to Notch proteins, a class of surface receptors poised for mechano-proteolytic activation, including an evolutionarily conserved mechanism of cleavage. However, so far there is no unifying explanation for why aGPCRs are autoproteolytically processed. This study introduces a genetically encoded sensor system to detect the dissociation events of aGPCR heterodimers into their constituent N-terminal and C-terminal fragments (NTFs and CTFs, respectively). An NTF release sensor (NRS) of the neural latrophilin-type aGPCR Cirl (ADGRL)(9-11), from Drosophila melanogaster, is stimulated by mechanical force. Cirl-NRS activation indicates that receptor dissociation occurs in neurons and cortex glial cells. The release of NTFs from cortex glial cells requires trans-interaction between Cirl and its ligand, the Toll-like receptor Tollo (Toll-8)(12), on neural progenitor cells, whereas expressing Cirl and Tollo in cis suppresses dissociation of the aGPCR. This interaction is necessary to control the size of the neuroblast pool in the central nervous system. It is concluded that receptor autoproteolysis enables non-cell-autonomous activities of aGPCRs, and that the dissociation of aGPCRs is controlled by their ligand expression profile and by mechanical force. The NRS system will be helpful in elucidating the physiological roles and signal modulators of aGPCRs, which constitute a large untapped reservoir of drug targets for cardiovascular, immune, neuropsychiatric and neoplastic diseases.
Koh, W. S., Knudsen, C., Izumikawa, T., Nakato, E., Grandt, K., Kinoshita-Toyoda, A., Toyoda, H. and Nakato, H. (2023). Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe. J Cell Sci 136(7). PubMed ID: 36897575
Summary:
Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. This study found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. This study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.
Huang, Z., Wang, W., Xu, P., Gong, S., Hu, Y., Liu, Y., Su, F., Anjum, K. M., Deng, W. M., Yang, S., Liu, J., Jiao, R. and Chen, J. (2023). Drosophila Ectoderm-expressed 4 modulates JAK/STAT pathway and protects flies against Drosophila C virus infection. Front Immunol 14: 1135625. PubMed ID: 36817462
Summary:
Sterile alpha and HEAT/Armadillo motif-containing protein (SARM) is conserved in evolution and negatively regulates TRIF-dependent Toll signaling in mammals. The SARM protein from Litopenaeus vannamei and its Drosophila orthologue Ectoderm-expressed (Ect4/SARM) are also involved in immune defense against pathogen infection. However, the functional mechanism of the protective effect remains unclear. This study shows that Ect4 is essential for the viral load in flies after a Drosophila C virus (DCV) infection. Viral load is increased in Ect4 mutants resulting in higher mortality rates than wild-type. Overexpression of Ect4 leads to a suppression of virus replication and thus improves the survival rate of the animals. Ect4 is required for the viral induction of STAT-responsive genes, TotA and TotM. Furthermore, Ect4 interacts with Stat92E, affecting the tyrosine phosphorylation and nuclear translocation of Stat92E in S2 cells. Altogether, this study identifies the adaptor protein Ect4 of the Toll pathway contributes to resistance to viral infection and regulates JAK/STAT signaling pathway.
Lai, Y. T., Sasamura, T., Kuroda, J., Maeda, R., Nakamura, M., Hatori, R., Ishibashi, T., Taniguchi, K., Ooike, M., Taguchi, T., Nakazawa, N., Hozumi, S., Okumura, T., Aigaki, T., Inaki, M. and Matsuno, K. (2023). The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis. Development 150(6). PubMed ID: 36861793
Summary:
Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. This study has identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.
Liao, Y. H., Wu, J. T., Hsieh, I. C., Lee, H. H. and Huang, P. H. (2023). ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress. iScience 26(2): 106005. PubMed ID: 36798436
Summary:
Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). This study established ARMS-knockdown in melanoma cells (siARMS) and in Drosophila eyes (GMR>dARMS (RNAi)) and challenged them with H(2)O(2). Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H(2)O(2)-triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in siARMS cells and GMR>dARMS (RNAi) fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. This study has thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress.

Friday, August 11th - Disease Models

Nemtsova, Y., Steinert, B. L. and Wharton, K. A. (2023). Compartment specific mitochondrial dysfunction in Drosophila knock-in model of ALS reversed by altered gene expression of OXPHOS subunits and pro-fission factor Drp1. Mol Cell Neurosci 125: 103834. PubMed ID: 36868541
Summary:
Amyotrophic Lateral Sclerosis (ALS) is a fatal multisystem neurodegenerative disease, characterized by a loss in motor function. ALS is genetically diverse, with mutations in genes ranging from those regulating RNA metabolism, like TAR DNA-binding protein (TDP-43) and Fused in sarcoma (FUS), to those that act to maintain cellular redox homeostasis, like superoxide dismutase 1 (SOD1). Although varied in genetic origin, pathogenic and clinical commonalities are clearly evident between cases of ALS. Defects in mitochondria is one such common pathology, thought to occur prior to, rather than as a consequence of symptom onset, making these organelles a promising therapeutic target for ALS, as well as other neurodegenerative diseases. Depending on the homeostatic needs of neurons throughout life, mitochondria are normally shuttled to different subcellular compartments to regulate metabolite and energy production, lipid metabolism, and buffer calcium. While originally considered a motor neuron disease due to the dramatic loss in motor function accompanied by motor neuron cell death in ALS patients, many studies have now implicated non-motor neurons and glial cells alike. Defects in non-motor neuron cell types often preceed motor neuron death suggesting their dysfunction may initiate and/or facilitate the decline in motor neuron health. This study investigate mitochondria in a Drosophila Sod1 knock-in model of ALS. In depth, in vivo, examination reveals mitochondrial dysfunction evident prior to onset of motor neuron degeneration. Genetically encoded redox biosensors identify a general disruption in the electron transport chain (ETC). Compartment specific abnormalities in mitochondrial morphology is observed in diseased sensory neurons, accompanied by no apparent defects in the axonal transport machinery, but instead an increase in mitophagy in synaptic regions. The decrease in networked mitochondria at the synapse is reversed upon downregulation of the pro-fission factor Drp1. Furthermore, altered expression of specific OXPHOS subunits reverses ALS-associated defects in mitochondrial morphology and function.
Maitra, U., Conger, J., Owens, M. M. M. and Ciesla, L. (2023). Predicting structural features of selected flavonoids responsible for neuroprotection in a Drosophila model of Parkinson's disease. Neurotoxicology 96: 1-12. PubMed ID: 36822376
Summary:
Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief, thereby underscoring the need for novel strategies to combat disease onset and progression. This study has employed an environmental toxin-induced Drosophila Parkinson's disease (PD) model as an inexpensive in vivo screening platform to explore the neuroprotective potential of selected dietary flavonoids. A specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes was indentified involving reduced survival, mobility defects, and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on this screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain α,β-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties through nuclear factor erythroid 2-related factor 2 (Nrf2) but also requires regulation of the immune deficiency (IMD) pathway involving NFκB and the negative regulator poor Imd response upon knock-in (Pirk). These data have identified specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions.
Lewis, S. A., Bakhtiari, S., Forstrom, J., Bayat, A., Bilan, F., Le Guyader, G., Alkhunaizi, E., Vernon, H., Padilla-Lopez, S. R. and Kruer, M. C. (2023). AGAP1-associated endolysosomal trafficking abnormalities link gene-environment interactions in a neurodevelopmental disorder. bioRxiv. PubMed ID: 36778426
Summary:
AGAP1 is an Arf1 GAP that regulates endolysosomal trafficking. Damaging variants have been linked to cerebral palsy and autism. This study reports 3 new individuals with microdeletion variants in AGAP1. Affected individuals have intellectual disability (3/3), autism (3/3), dystonia with axial hypotonia (1/3), abnormalities of brain maturation (1/3), growth impairment (2/3) and facial dysmorphism (2/3). Mechanisms potentially underlying AGAP1 neurodevelopmental impairments were investigated using the Drosophila ortholog, CenG1a. Reduced axon terminal size, increased neuronal endosome abundance, and elevated autophagy were discovered at baseline. Given potential incomplete penetrance, gene-environment interactions were assessed. Basal elevation in phosphorylation of the integrated stress-response protein eIF2α and inability to further increase eIF2α-P with subsequent cytotoxic stressors was found. CenG1a -mutant flies have increased lethality from exposure to environmental insults. A model is proposed wherein disruption of AGAP1 function impairs endolysosomal trafficking, chronically activating the integrated stress response, and leaving AGAP1-deficient cells susceptible to a variety of second hit cytotoxic stressors. This model may have broader applicability beyond AGAP1 in instances where both genetic and environmental insults co-occur in individuals with neurodevelopmental disorders. This study has described 3 additional patients with heterozygous AGAP1 deletion variants and used a loss of function Drosophila model to identify defects in synaptic morphology with increased endosomal sequestration, chronic autophagy induction, basal activation of eIF2α-P, and sensitivity to environmental stressors.
Odenthal, J., Dittrich, S., Ludwig, V., Merz, T., Reitmeier, K., Reusch, B., H0hne, M., Cosgun, Z. C., Hohenadel, M., Putnik, J., Gobel, H., Rinschen, M. M., Altmuller, J., Koehler, S., Schermer, B., Benzing, T., Beck, B. B., Brinkkotter, P. T., Habbig, S. and Bartram, M. P. (2023). Modeling of ACTN4-Based Podocytopathy Using Drosophila Nephrocytes. Kidney Int Rep 8(2): 317-329. PubMed ID: 36815115
Summary:
Genetic disorders are among the most prevalent causes leading to progressive glomerular disease and, ultimately, end-stage renal disease (ESRD) in children and adolescents. Identification of underlying genetic causes is indispensable for targeted treatment strategies and counseling of affected patients and their families. This study reports on a boy who presented at 4 years of age with proteinuria and biopsy-proven focal segmental glomerulosclerosis (FSGS) that was temporarily responsive to treatment with ciclosporin A. Molecular genetic testing identified a novel mutation in alpha-actinin-4 (p.M240T). A feasible and efficient experimental approach is descibed to test its pathogenicity by combining in silico, in vitro, and in vivo analyses. The de novo p.M240T mutation led to decreased alpha-actinin-4 stability as well as protein mislocalization and actin cytoskeleton rearrangements. Transgenic expression of wild-type human alpha-actinin-4 in Drosophila melanogaster nephrocytes was able to ameliorate phenotypes associated with the knockdown of endogenous actinin. In contrast, p.M240T, as well as other established disease variants p.W59R and p.K255E, failed to rescue these phenotypes, underlining the pathogenicity of the novel alpha-actinin-4 variant. These data highlight that the newly identified alpha-actinin-4 mutation indeed encodes for a disease-causing variant of the protein and promote the Drosophila model as a simple and convenient tool to study monogenic kidney disease in vivo.
Li, Y., Geng, J., Rimal, S., Wang, H., Liu, X., Lu, B. and Li, S. (2023). The mTORC2/AKT/VCP axis is associated with quality control of the stalled translation of poly(GR) dipeptide repeats in C9-ALS/FTD. J Biol Chem 299(3): 102995. PubMed ID: 36764521
Summary:
Expansion of G4C2 hexanucleotide repeats in the chromosome 9 ORF 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (C9-ALS/FTD). Dipeptide repeats generated by unconventional translation, especially the R-containing poly(GR), have been implicated in C9-ALS/FTD pathogenesis. Mutations in other genes, including TAR DNA-binding protein 43 KD (TDP-43), fused in sarcoma (FUS), and valosin-containing protein, have also been linked to ALS/FTD, and upregulation of amyloid precursor protein (APP) is observed at the early stage of ALS and FTD. Fundamental questions remain as to the relationships between these ALS/FTD genes and whether they converge on similar cellular pathways. In this study, using biochemical, cell biological, and genetic analyses in Drosophila disease models, patient-derived fibroblasts, and mammalian cell culture, it was shown that mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling is activated by APP, TDP-43, and FUS and that mTORC2/AKT and its downstream target valosin-containing protein mediate the effect of APP, TDP-43, and FUS on the quality control of C9-ALS/FTD-associated poly(GR) translation. This study also found that poly(GR) expression results in reduction of global translation and that the coexpression of APP, TDP-43, and FUS results in further reduction of global translation, presumably through the GCN2/eIF2α-integrated stress response pathway. Together, these results implicate mTORC2/AKT signaling and GCN2/eIF2α-integrated stress response as common signaling pathways underlying ALS/FTD pathogenesis.
Larnerd, C., Adhikari, P., Valdez, A., Del Toro, A. and Wolf, F. W. (2023). Rapid and Chronic Ethanol Tolerance Are Composed of Distinct Memory-Like States in Drosophila. J Neurosci 43(12): 2210-2220. PubMed ID: 36750369
Summary:
Ethanol tolerance is the first type of behavioral plasticity and neural plasticity that is induced by ethanol intake, and yet its molecular and circuit bases remain largely unexplored. This study characterize the following three distinct forms of ethanol tolerance in male Drosophila: rapid, chronic, and repeated. Rapid tolerance is composed of two short-lived memory-like states, one that is labile and one that is consolidated. Chronic tolerance, induced by continuous exposure, lasts for 2 d, induces ethanol preference, and hinders the development of rapid tolerance through the activity of histone deacetylases (HDACs). Unlike rapid tolerance, chronic tolerance is independent of the immediate early gene Hr38/Nr4a Chronic tolerance is suppressed by the sirtuin HDAC Sirt1, whereas rapid tolerance is enhanced by Sirt1. Moreover, rapid and chronic tolerance map to anatomically distinct regions of the mushroom body learning and memory centers. Chronic tolerance, like long-term memory, is dependent on new protein synthesis and it induces the kayak/c-fos immediate early gene, but it depends on CREB signaling outside the mushroom bodies, and it does not require the Radish GTPase. Thus, chronic ethanol exposure creates an ethanol-specific memory-like state that is molecularly and anatomically different from other forms of ethanol tolerance.

Thursday, August 10th - RNAs and Transposons

Miller, D. E., Dorador, A. P, ..., Hawley, R. S. and Blumenstiel, J. P. (2023). Off-target piRNA gene silencing in Drosophila melanogaster rescued by a transposable element insertion. PLoS Genet 19(2): e1010598. PubMed ID: 36809339
Summary:
Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. This study describes how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. This local gene silencing occurs in cis and is dependent on deadlock, a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. It was further shown how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex.
Mercier, J., Nagengast, A. A. and DiAngelo, J. R. (2023). The role of SR protein kinases in regulating lipid storage in the Drosophila fat body. Biochem Biophys Res Commun 649: 10-15. PubMed ID: 36738578
Summary:
The survival of animals during periods of limited nutrients is dependent on the organism's ability to store lipids during times of nutrient abundance. However, the increased availability of food in modern western society has led to an excess storage of lipids resulting in metabolic diseases. To better understand the genes involved in regulating lipid storage, genome-wide RNAi screens were performed in cultured Drosophila cells and one group of genes identified includes mRNA splicing factor genes. A group of splicing factors important for intron/exon border recognition known as SR proteins are involved in controlling lipid storage in Drosophila; however, how these SR proteins are regulated to control lipid storage is not fully understood. This study focussed on two SR protein kinases (SRPKs) in Drosophila: SRPK and SRPK79D. Decreasing the expression of these genes specifically in the adult fat body using RNAi resulted in lower levels of triglycerides and this is due to a decrease in the amount of fat stored per cell, despite having more fat cells, when compared to control flies. Decreasing SRPK and SRPK79D levels in the fat body leads to altered splicing of the β-oxidation gene, carnitine palmitoyltransferase 1 (CPT1), resulting in increased production of a more active enzyme, which would increase lipid breakdown and be consistent with the lean phenotype observed in these flies. In addition, flies with decreased SRPK and SRPK79D levels in their fat bodies eat less, which may also contribute to the decreased triglyceride phenotype. Together, these findings provide evidence to support that lipid storage is controlled by the phosphorylation of factors involved in mRNA splicing.
Pekovic, F., Rammelt, C., Kubikova, J., Metz, J., Jeske, M. and Wahle, E. (2023). RNA binding proteins Smaug and Cup induce CCR4-NOT-dependent deadenylation of the nanos mRNA in a reconstituted system. Nucleic Acids Res. PubMed ID: 36951092
Summary:
Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior - posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug, which binds to Smaug recognition elements (SREs) in the nanos 3'-UTR and nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. This study reports an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has a minor inhibitory effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.
Xu, Q., Liu, J., Du, X., Xue, D., Li, D. and Bi, X. (2023). Long non-coding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila. Genetics. PubMed ID: 36930573
Summary:
Long non-coding RNAs (lncRNAs) play important regulatory roles in stem cells self-renewal, pluripotency maintenance and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISCs regeneration in Drosophila have yet been characterized. This study identify a lncRNA, CR46040, that is essential for the injury-induced ISCs regeneration in Drosophila. Loss of CR46040 greatly impairs ISCs proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. This study demonstrates that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site, and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies are failed to fully activate JNK, JAK/STAT and HIPPO signaling pathways after tissue damage, which are required for ISCs proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. These findings characterize, for the first time, a lncRNA that mediates damage-induced ISCs proliferation in Drosophila, and provide new insights into the functional links among the long non-coding RNAs, ISCs proliferation and tissue homeostasis.
Zhang, Z., Bae, B., Cuddleston, W. H. and Miura, P. (2023). Coordination of Alternative Splicing and Alternative Polyadenylation revealed by Targeted Long-Read Sequencing. bioRxiv. PubMed ID: 36993601
Summary:
Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. In this study, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach called Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3' ends. Using PL-Seq, genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs were identified. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.
Siddiqui, N. U., Karaiskakis, A., Goldman, A. L., Eagle, W. V. I., Smibert, C. A., Gavis, E. R. and Lipshitz, H. D. (2023). Smaug regulates germ plasm synthesis and primordial germ cell number in Drosophila embryos by repressing the oskar and bruno 1 mRNAs. bioRxiv. PubMed ID: 36909513 :
Summary:
During Drosophila oogenesis, the Oskar (Osk) RNA-binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. This study identified the mechanisms that regulate the osk mRNA in the early embryo. The Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk mRNA itself as well as the bruno 1 (bru1) mRNA, which encodes an RBP that promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNAs results in ectopic translation of these transcripts in the germ plasm and excess PGCs. SMG therefore triggers a post-transcriptional regulatory pathway that attenuates germ plasm synthesis in embryos, thus modulating the number of PGCs.

Wednesday, August 9th - Stem Cells

Ayachit, M. S. and Shravage, B. V. (2023). Atg1 modulates mitochondrial dynamics to promote germline stem cell maintenance in Drosophila. Biochem Biophys Res Commun 643: 192-202. PubMed ID: 36621115
Summary:
Mitochondrial dynamics (fusion and fission) are necessary for stem cell maintenance and differentiation. However, the relationship between mitophagy, mitochondrial dynamics and stem cell exhaustion needs to be clearly understood. This study reports the multifaceted role of Atg1 in mitophagy, mitochondrial dynamics and stem cell maintenance in female germline stem cells (GSCs) in Drosophila.Depletion of Atg1 in GSCs leads to impaired autophagy and mitophagy as measured by reduced formation of autophagosomes, increased accumulation of p62/Ref (2)P and accumulation of damaged mitochondria. Disrupting Atg1 function led to mitochondrial fusion in developing cysts. The fusion resulted from an increase in Marf levels in both GSCs and cysts, and the fusion phenotype could be rescued by overexpression of Drp1 or by depleting Marf via RNAi in Atg1-depleted cyst cells. Interestingly, double knockdown of both Atg1:Drp1 led to the significant loss of germ cells (GCs) as compared to Atg1KD and Drp1KD. Strikingly, Atg1:Marf double knockdown leads to a dramatic loss of GSCs, GCs and a total loss of vitellogenic stages, suggesting a block in oogenesis. Overall, these results demonstrate that Drp1, Marf and Atg1 function together to influence female GSC maintenance, their differentiation into cysts and oogenesis in Drosophila.
Meghini, F., Martins, T., Zhang, Q., Loyer, N., Trickey, M., Abula, Y., Yamano, H., Januschke, J. and Kimata, Y. (2023). APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells. EMBO Rep 24(4): e55607. PubMed ID: 36852890
Summary:
A functional centrosome is vital for the development and physiology of animals. Among numerous regulatory mechanisms of the centrosome, ubiquitin-mediated proteolysis is known to be critical for the precise regulation of centriole duplication. However, its significance beyond centrosome copy number control remains unclear. Using an in vitro screen for centrosomal substrates of the APC/C ubiquitin ligase in Drosophila, several conserved pericentriolar material (PCM) components were identified, including the inner PCM protein Spd2. W Spd2 levels are controlled by the interphase-specific form of APC/C, APC/C(Fzr), in cultured cells and developing brains. Increased Spd2 levels compromise neural stem cell-specific asymmetric PCM recruitment and microtubule nucleation at interphase centrosomes, resulting in partial randomisation of the division axis and segregation patterns of the daughter centrosome in the following mitosis. Evidencse is provided that APC/C(Fzr) -dependent Spd2 degradation restricts the amount and mobility of Spd2 at the daughter centrosome, thereby facilitating the accumulation of Polo-dependent Spd2 phosphorylation for PCM recruitment. This study underpins the critical role of cell cycle-dependent proteolytic regulation of the PCM in stem cells.
Wu, S., Yang, Y., Tang, R., Zhang, S., Qin, P., Lin, R., Rafel, N., Lucchetta, E. M., Ohlstein, B. and Guo, Z. (2023). Apical-basal polarity precisely determines intestinal stem cell number by regulating Prospero threshold. Cell Rep 42(2): 112093. PubMed ID: 36773292
Summary:
Apical-basal polarity and cell-fate determinants are crucial for the cell fate and control of stem cell numbers. However, their interplay leading to a precise stem cell number remains unclear. Drosophila pupal intestinal stem cells (pISCs) asymmetrically divide, generating one apical ISC progenitor and one basal Prospero (Pros)(+) enteroendocrine mother cell (EMC), followed by symmetric divisions of each daughter before adulthood, providing an ideal system to investigate the outcomes of polarity loss. Using lineage tracing and ex vivo live imaging, this study identified an interlocked polarity regulation network precisely determining ISC number: Bazooka inhibits Pros accumulation by activating Notch signaling to maintain stem cell fate in pISC apical daughters. A threshold of Pros promotes differentiation to EMCs and avoids ISC-like cell fate, and over-threshold of Pros inhibits miranda expression to ensure symmetric divisions in pISC basal daughters. This work suggests that a polarity-dependent threshold of a differentiation factor precisely controls stem cell number.
Sun, H., Shah, A. S., Bonfini, A., Buchon, N. S. and Baskin, J. M. (2023). Wnt/beta-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation. bioRxiv. PubMed ID: 36865263
Summary:
The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/beta-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, \the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation, and kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/beta-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling.
Raz, A. A., Vida, G. S., Stern, S. R., ..., Krause, H., Matunis, E. L., White-Cooper, H., DiNardo, S. and Fuller, M. T. (2023). Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes. Elife 12. PubMed ID: 36795469
Summary:
This study provides a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. Assignment of key germline and somatic cell types was assigned using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, datasets compatible with commonly used software such as Seurat and Monocle is provided. The foundation provided in this study will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.
Tian, A., Morejon, V., Kohoutek, S., Huang, Y. C., Deng, W. M. and Jiang, J. (2022). Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut. Embo J: e110834. PubMed ID: 35950466
Summary:
Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. This study shows that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. Ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, the dividing enteroblasts did not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, this work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.

Tuesday, August 8th - Adult Neural Structure, Development and Function

Mayseless, O., Shapira, G., Rachad, E. Y., Fiala, A. and Schuldiner, O. (2023). Neuronal excitability as a regulator of circuit remodeling. Curr Biol 33(5): 981-989.e983. PubMed ID: 36758544
Summary:
Postnatal remodeling of neuronal connectivity shapes mature nervous systems. The pruning of exuberant connections involves cell-autonomous and non-cell-autonomous mechanisms, such as neuronal activity. Indeed, experience-dependent competition sculpts various excitatory neuronal circuits. Moreover, activity has been shown to regulate growth cone motility and the stability of neurites and synaptic connections. However, whether inhibitory activity influences the remodeling of neuronal connectivity or how activity influences remodeling in systems in which competition is not clearly apparent is not fully understood. This study used the Drosophila mushroom body (MB) as a model to examine the role of neuronal activity in the developmental axon pruning of γ-Kenyon cells. The MB is a neuronal structure in insects, implicated in associative learning and memory, which receives mostly olfactory input from the antennal lobe. The MB circuit includes intrinsic neurons, called Kenyon cells (KCs), which receive inhibitory input from the GABAergic anterior paired lateral (APL) neuron among other inputs. The γ-KCs undergo stereotypic, steroid-hormone-dependent remodeling( that involves the pruning of larval neurites followed by regrowth to form adult connections. This study demonstrates that silencing neuronal activity is required for γ-KC pruning. Furthermore, it was shown that this is mechanistically achieved by cell-autonomous expression of the inward rectifying potassium channel 1 (irk1) combined with inhibition by APL neuron activity likely via GABA-B-R1 signaling. These results support the Hebbian-like rule "use it or lose it," where inhibition can destabilize connectivity and promote pruning while excitability stabilizes existing connections.
Mishra, A., Serbe-Kamp, E., Borst, A. and Haag, J. (2023). Voltage to Calcium Transformation Enhances Direction Selectivity in Drosophila T4 Neurons. J Neurosci 43(14): 2497-2514. PubMed ID: 36849417
Summary:
An important step in neural information processing is the transformation of membrane voltage into calcium signals leading to transmitter release. However, the effect of voltage to calcium transformation on neural responses to different sensory stimuli is not well understood. This study used in vivo two-photon imaging of genetically encoded voltage and calcium indicators, ArcLight and GCaMP6f, respectively, to measure responses in direction-selective T4 neurons of female Drosophila Comparison between ArcLight and GCaMP6f signals reveals calcium signals to have a significantly higher direction selectivity compared with voltage signals. Using these recordings, a model was built which transforms T4 voltage responses into calcium responses. Using a cascade of thresholding, temporal filtering and a stationary nonlinearity, the model reproduces experimentally measured calcium responses across different visual stimuli. These findings provide a mechanistic underpinning of the voltage to calcium transformation and show how this processing step, in addition to synaptic mechanisms on the dendrites of T4 cells, enhances direction selectivity in the output signal of T4 neurons. Measuring the directional tuning of postsynaptic vertical system (VS)-cells with inputs from other cells blocked, this study found that, indeed, it matches the one of the calcium signal in presynaptic T4 cells.
Lyu, S., Terao, N., Nakashima, H., Itoh, M. and Tonoki, A. (2023). Neuropeptide diuretic hormone 31 mediates memory and sleep via distinct neural pathways in Drosophila. Neurosci Res. PubMed ID: 36780946
Summary:
Memory formation and sleep regulation are critical for brain functions in animals from invertebrates to humans. Neuropeptides play a pivotal role in regulating physiological behaviors, including memory formation and sleep. However, the detailed mechanisms by which neuropeptides regulate these physiological behaviors remains unclear. This study report sthat neuropeptide diuretic hormone 31 (DH31) positively regulates memory formation and sleep in Drosophila melanogaster. The expression of DH31 in the dorsal and ventral fan-shaped body (dFB and vFB) neurons of the central complex and ventral lateral clock neurons (LNvs) in the brain was responsive to sleep regulation. In addition, the expression of membrane-tethered DH31 in dFB neurons rescued sleep defects in Dh31 mutants, suggesting that DH31 secreted from dFB, vFB, and LNvs acts on the DH31 receptor in the dFB to regulate sleep partly in an autoregulatory feedback loop. Moreover, the expression of DH31 in octopaminergic neurons, but not in the dFB neurons, is involved in forming intermediate-term memory. These results suggest that DH31 regulates memory formation and sleep through distinct neural pathways.
Marmor-Kollet, N., Berkun, V., Cummings, G., Keren-Shaul, H., David, E., Addadi, Y. and Schuldiner, O. (2023). Actin-dependent astrocytic infiltration is a key step for axon defasciculation during remodeling. Cell Rep 42(2): 112117. PubMed ID: 36790930
Summary:
Astrocytes are essential for synapse formation, maturation, and plasticity; however, their function during developmental neuronal remodeling is largely unknown. To identify astrocytic molecules required for axon pruning of mushroom body (MB) γ neurons in Drosophila, astrocytes were profiled before (larva) and after (adult) remodeling. Focusing on genes enriched in larval astrocytes, 12 astrocytic genes were identified that are required for axon pruning, including the F-actin regulators Actin-related protein 2/3 complex, subunit 1 (Arpc1) and formin3 (form3). Interestingly, perturbing astrocytic actin dynamics does not affect their gross morphology, migration, or transforming growth factorβ (TGF-β) secretion. In contrast, actin dynamics is required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitates infiltration by Arpc1 knockdown (KD) astrocytes and promotes axon pruning. Conversely, increased axonal adhesion reduces lobe infiltration by wild-type (WT) astrocytes. Together, these findings suggest that actin-dependent astrocytic infiltration is a key step in axon pruning, thus promoting understanding of neuron-glia interactions during remodeling.
Mabuchi, Y., Cui, X., Xie, L., Kim, H., Jiang, T. and Yapici, N. (2023). GABA-mediated inhibition in visual feedback neurons fine-tunes Drosophila male courtship. bioRxiv. PubMed ID: 36747836
Summary:
Vision is critical for the regulation of mating behaviors in many species. This study discovered that the Drosophila ortholog of human GABA (A) -receptor-associated protein (GABARAP; Atg8a) is required to fine-tune male courtship by modulating the activity of visual feedback neurons, lamina tangential cells (Lat). GABARAP is a ubiquitin-like protein that regulates cell-surface levels of GABA (A) receptors. Knocking down GABARAP or GABA (A) receptors in Lat neurons or hyperactivating them induces male courtship toward other males. Inhibiting Lat neurons, on the other hand, delays copulation by impairing the ability of males to follow females. Remarkably, the human ortholog of Drosophila GABARAP restores function in Lat neurons. Using in vivo two-photon imaging and optogenetics, Lat neurons were shown to be functionally connected to neural circuits that mediate visually-guided courtship pursuits in males. This work reveals a novel physiological role for GABARAP in fine-tuning the activity of a visual circuit that tracks a mating partner during courtship.
Ma, D., Herndon, N., Le, J. Q., Abruzzi, K. C., Zinn, K. and Rosbash, M. (2023). Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in the Drosophila adult brain. Sci Adv 9(8): eade8500. PubMed ID: 36812309
Summary:
Recent single-cell sequencing of most adult Drosophila circadian neurons indicated notable and unexpected heterogeneity. To address whether other populations are similar, a large subset of adult brain dopaminergic neurons was sequenced. Their gene expression heterogeneity is similar to that of clock neurons, i.e., both populations have two to three cells per neuron group. There was also unexpected cell-specific expression of neuron communication molecule messenger RNAs: G protein-coupled receptor or cell surface molecule (CSM) transcripts alone can define adult brain dopaminergic and circadian neuron cell type. Moreover, the adult expression of the CSM DIP-beta in a small group of clock neurons is important for sleep. It is suggested that the common features of circadian and dopaminergic neurons are general, essential for neuronal identity and connectivity of the adult brain, and that these features underlie the complex behavioral repertoire of Drosophila.

Monday, August 7th - Adult Physiology and Metabolism

Liu, Z., Pan, X., Guo, J., Li, L., Tang, Y., Wu, G., Li, M. and Wang, H. (2023). Long-term sevoflurane exposure resulted in temporary rather than lasting cognitive impairment in Drosophila. Behav Brain Res 442: 114327. PubMed ID: 36738841
Summary:
Sevoflurane is the primary inhaled anesthetic used in pediatric surgery. It has been the focus of research since animal models studies found that it was neurotoxic to the developing brain two decades ago. However, whether pediatric general anesthesia can lead to permanent cognitive deficits remained a subject of heated debate. Therefore, this study aims to determine the lifetime neurotoxicity of early long-time sevoflurane exposure using a short-life-cycle animal model, Drosophila melanogaster. To investigate this question, the lifetime changes of two-day-old flies' learning and memory abilities after anesthesia with 3 % sevoflurane for 6 h by the T-maze memory assay. Apoptosis, levels of ATP and ROS, and related genes were evaluated in the fly head. The results suggest that 6 h 3 % sevoflurane exposure at a young age can only induce transient neuroapoptosis and cognitive deficits around the first week after anesthesia. But this brain damage recedes with time and vanishes in late life. It was also found that the mRNA level of caspases and Bcl-2, ROS level, and ATP level increased during this temporary neuroapoptosis process. And mRNA levels of antioxidants, such as SOD2 and CAT, increased and decreased simultaneously with the rise and fall of the ROS level, indicating a possible contribution to the recovery from the sevoflurane impairment. In conclusion, these results suggest that one early prolonged sevoflurane-based general anesthesia can induce neuroapoptosis and learning and memory deficit transiently but not permanently in Drosophila.
Li, H., Luo, X., Li, N., Liu, T. and Zhang, J. (2023). Insulin-like peptide 8 (Ilp8) regulates female fecundity in flies. Front Cell Dev Biol 11: 1103923. PubMed ID: 36743416
Summary:
Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. This study reports the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster. Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Drosophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis. Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 is specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly. These findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8.
Li, A. Q., Li, S. S., Zhang, R. X., Zhao, X. Y., Liu, Z. Y., Hu, Y., Wang, B., Neely, G. G., Simpson, S. J. and Wang, Q. P. (2023). Nutritional geometry framework of sucrose taste in Drosophila. J Genet Genomics. PubMed ID: 36773723
Summary:
Dietary protein (P) and carbohydrate (C) have a major impact on sweet taste sensation. However, it remains unclear whether the balance of P and C influences sweet taste sensitivity. This study use the nutritional geometry framework (NGF) to address the interaction of protein and carbohydrates and on sweet taste using Drosophila as a model. The results reveal that high-protein, low-carbohydrate (HPLC) diets sensitize to sweet taste and low-protein, high-carbohydrate (LPHC) diets desensitize sweet taste in both male and female flies. The underlying mechanisms of these two diets' effect on sweet taste was further investigated using RNA sequencing. When compared to the LPHC diet, the mRNA expression of genes involved in the metabolism of glycine, serine, and threonine is significantly upregulated in the HPLC diet, suggesting these amino acids may mediate sweet taste perception. It was further found that sweet sensitization occurs in flies fed with the LPHC diet supplemented with serine and threonine. This study demonstrates that sucrose taste sensitivity is affected by the balance of dietary protein and carbohydrates possibly through changes in serine and threonine.
Li, Q., Wang, L., Cao, Y., Wang, X., Tang, C. and Zheng, L. (2023). Stable Expression of dmiR-283 in the Brain Promises Positive Effects in Endurance Exercise on Sleep-Wake Behavior in Aging Drosophila. Int J Mol Sci 24(4). PubMed ID: 36835595
Summary:
Sleep-wake stability is imbalanced with natural aging, and microRNAs (miRNAs) play important roles in cell proliferation, apoptosis, and aging; however, the biological functions of miRNAs in regulating aging-related sleep-wake behavior remain unexplored. This study varied the expression pattern of dmiR-283 in Drosophila and the result showed that the aging decline in sleep-wake behavior was caused by the accumulation of brain dmiR-283 expression, whereas the core clock genes cwo and Notch signaling pathway might be suppressed, which regulate the aging process. In addition, to identify exercise intervention programs of Drosophila that promote healthy aging, mir-283SP/+ (mir-23SP referes to mir-283sponge) and Pdf > mir-283SP flies were driven to perform endurance exercise for a duration of 3 weeks starting at 10 and 30 days, respectively. The results showed that exercise starting in youth leads to an enhanced amplitude of sleep-wake rhythms, stable periods, increased activity frequency upon awakening, and the suppression of aging brain dmiR-283 expression in mir-283SP/+ middle-aged flies. Conversely, exercise performed when the brain dmiR-283 reached a certain accumulation level showed ineffective or negative effects. In conclusion, the accumulation of dmiR-283 expression in the brain induced an age-dependent decline in sleep-wake behavior. Endurance exercise commencing in youth counteracts the increase in dmiR-283 in the aging brain, which ameliorates the deterioration of sleep-wake behavior during aging.
Livelo, C., Guo, Y., Abou Daya, F., Rajasekaran, V., Varshney, S., Le, H. D., Barnes, S., Panda, S. and Melkani, G. C. (2023). Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models. Nat Commun 14(1): 949. PubMed ID: 36810287
Summary:
Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. This study demonstrates that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, these data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments.
Silva, E. A. B., Venda, A. M. and Homem, C. C. F. (2023). Serine hydroxymethyl transferase (Shmt) is required for optic lobe neuroepithelia development in Drosophila. Development. PubMed ID: 36896963
Summary:
Cell fate and growth require one-carbon units for the biosynthesis of nucleotides, methylation reactions, and redox homeostasis, provided by one-carbon metabolism. Consistently, defects in one-carbon metabolism lead to severe developmental defects, such as neural tube defects. However, the role of this pathway during brain development and in neural stem cell regulation is poorly understood. To better understand the role of one carbon metabolism this study focused on the enzyme Serine hydroxymethyl transferase (Shmt), a key player of the one-carbon cycle, during Drosophila brain development. Although loss of shmt does not cause obvious defects in the central brain, it leads to severe phenotypes in the optic lobe. The shmt mutants have smaller optic lobe neuroepithelia partly justified by increased apoptosis. Additionally, shmt mutant neuroepithelia have morphological defects, failing to form a lamina furrow likely explaining the observed absence of lamina neurons. These findings show that one-carbon metabolism is critical for the normal development of neuroepithelia, and consequently for the generation of neural progenitor cells and neurons. These results propose a mechanistic role for one-carbon during brain development.

Friday, August 4th - Adult neural development and function

Frame, A. K., Robinson, J. W., Mahmoudzadeh, N. H., Tennessen, J. M., Simon, A. F. and Cumming, R. C. (2023). Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies. Aging (Albany NY) 15(4): 947-981. PubMed ID: 36849157
Summary:
The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. This study genetically manipulated expression of Drosophila melanogaster Lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. Survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites were also examined. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. Evidence is provided that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, these findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner.
Kuwano, R., Katsura, M., Iwata, M., Yokosako, T. and Yoshii, T. (2023). Pigment-dispersing factor and CCHamide1 in the Drosophila circadian clock network. Chronobiol Int: 1-16. PubMed ID: 36786215
Summary:
Animals possess a circadian central clock in the brain, where circadian behavioural rhythms are generated. In the fruit fly (Drosophila melanogaster), the central clock comprises a network of approximately 150 clock neurons, which is important for the maintenance of a coherent and robust rhythm. Several neuropeptides involved in the network have been identified, including Pigment-dispersing factor (PDF) and CCHamide1 (CCHa1) neuropeptides. PDF signals bidirectionally to CCHa1-positive clock neurons; thus, the clock neuron groups expressing PDF and CCHa1 interact reciprocally. However, the role of these interactions in molecular and behavioural rhythms remains elusive. This study,generated Pdf01 and CCHa1SK8) double mutants and examined their locomotor activity-related rhythms. The single mutants of Pdf01 or CCHa1SK8 displayed free-running rhythms under constant dark conditions, whereas approximately 98% of the double mutants were arrhythmic. In light-dark conditions, the evening activity of the double mutants was phase-advanced compared with that of the single mutants. In contrast, both the single and double mutants had diminished morning activity. These results suggest that the effects of the double mutation varied in behavioural parameters. The double and triple mutants of per 01, Pdf01, and CCHa1SK8 further revealed that PDF signalling plays a role in the suppression of activity during the daytime under a clock-less background. These results provide insights into the interactions between PDF and CCHa1 signalling and their roles in activity rhythms.
Ellis, K. E., Smihula, H., Ganguly, I., Vigato, E., Bervoets, S., Auer, T. O., Benton, R., Litwin-Kumar, A. and Caron, S. J. C. (2023). Evolution of connectivity architecture in the Drosophila mushroom body. bioRxiv. PubMed ID: 36798335
Summary:
Brain evolution has primarily been studied at the macroscopic level by comparing the relative size of homologous brain centers between species. How neuronal circuits change at the cellular level over evolutionary time remains largely unanswered. Using a phylogenetically informed framework, this study compared the olfactory circuits of three closely related Drosophila species that differ radically in their chemical ecology: the generalists Drosophila melanogaster and Drosophila simulans that feed on fermenting fruit, and Drosophila sechellia that specializes on ripe noni fruit. A central part of the olfactory circuit was examined that has not yet been investigated in these species - the connections between the projection neurons of the antennal lobe and the Kenyon cells of the mushroom body, an associative brain center - to identify species-specific connectivity patterns. Neurons encoding food odors - the DC3 neurons in D. melanogaster and D. simulans and the DL2d neurons in D. sechellia - connect more frequently with Kenyon cells, giving rise to species-specific biases in connectivity. These species-specific differences in connectivity reflect two distinct neuronal phenotypes: in the number of projection neurons or in the number of presynaptic boutons formed by individual projection neurons. This study shows how fine-grained aspects of connectivity architecture in a higher brain center can change during evolution to reflect the chemical ecology of a species.
Hertzler, J. I., Bernard, A. R. and Rolls, M. M. (2023). Dendrite regeneration mediates functional recovery after complete dendrite removal. Dev Biol 497: 18-25. PubMed ID: 36870669
Summary:
Unlike many cell types, neurons are not typically replaced if damaged. Therefore, regeneration of damaged cellular domains is critical for maintenance of neuronal function. While axon regeneration has been documented for several hundred years, it has only recently become possible to determine whether neurons respond to dendrite removal with regeneration. Regrowth of dendrite arbors has been documented in invertebrate and vertebrate model systems, but whether it leads to functional restoration of a circuit remains unknown. To test whether dendrite regeneration restores function, larval Drosophila nociceptive neurons were used. Their dendrites detect noxious stimuli to initiate escape behavior. Previous studies of Drosophila sensory neurons have shown that dendrites of single neurons regrow after laser severing. This studybremoved dendrites from 16 neurons per animal to clear most of the dorsal surface of nociceptive innervation. As expected, this reduced aversive responses to noxious touch. Surprisingly, behavior was completely restored 24 ​h after injury, at the stage when dendrite regeneration has begun, but the new arbor has only covered a small portion of its former territory. This behavioral recovery required regenerative outgrowth as it was eliminated in a genetic background in which new growth is blocked. It is concluded that dendrite regeneration can restore behavior.
Jones, J. D., Holder, B. L., Eiken, K. R., Vogt, A., Velarde, A. I., Elder, A. J., McEllin, J. A. and Dissel, S. (2023). Regulation of sleep by cholinergic neurons located outside the central brain in Drosophila. PLoS Biol 21(3): e3002012. PubMed ID: 36862736
Summary:
Sleep is a complex and plastic behavior regulated by multiple brain regions and influenced by numerous internal and external stimuli. Thus, to fully uncover the function(s) of sleep, cellular resolution of sleep-regulating neurons needs to be achieved. Doing so will help to unequivocally assign a role or function to a given neuron or group of neurons in sleep behavior. In the Drosophila brain, neurons projecting to the dorsal fan-shaped body (dFB) have emerged as a key sleep-regulating area. To dissect the contribution of individual dFB neurons to sleep, this study undertook an intersectional Split-GAL4 genetic screen focusing on cells contained within the 23E10-GAL4 driver, the most widely used tool to manipulate dFB neurons. This study demonstrated that 23E10-GAL4 expresses in neurons outside the dFB and in the fly equivalent of the spinal cord, the ventral nerve cord (VNC). Furthermore, it was shown that 2 VNC cholinergic neurons strongly contribute to the sleep-promoting capacity of the 23E10-GAL4 driver under baseline conditions. However, in contrast to other 23E10-GAL4 neurons, silencing these VNC cells does not block sleep homeostasis. Thus, these data demonstrate that the 23E10-GAL4 driver contains at least 2 different types of sleep-regulating neurons controlling distinct aspects of sleep behavior.
Lipshutz, D., Kashalikar, A., Farashahi, S. and Chklovskii, D. B. (2023). A linear discriminant analysis model of imbalanced associative learning in the mushroom body compartment. PLoS Comput Biol 19(2): e1010864. PubMed ID: 36745688
Summary:
To adapt to their environments, animals learn associations between sensory stimuli and unconditioned stimuli. In invertebrates, olfactory associative learning primarily occurs in the mushroom body, which is segregated into separate compartments. Within each compartment, Kenyon cells (KCs) encoding sparse odor representations project onto mushroom body output neurons (MBONs) whose outputs guide behavior. Associated with each compartment is a dopamine neuron (DAN) that modulates plasticity of the KC-MBON synapses within the compartment. Interestingly, DAN-induced plasticity of the KC-MBON synapse is imbalanced in the sense that it only weakens the synapse and is temporally sparse. This study proposes a normative mechanistic model of the MBON as a linear discriminant analysis (LDA) classifier that predicts the presence of an unconditioned stimulus (class identity) given a KC odor representation (feature vector). Starting from a principled LDA objective function and under the assumption of temporally sparse DAN activity, an online algorithm was derived that maps onto the mushroom body compartment. This model accounts for the imbalanced learning at the KC-MBON synapse and makes testable predictions that provide clear contrasts with existing models.

Thursday, August 3rd - Chromatin, DNA replication, and chromosome dynamics

Jauregui-Lozano, J., McGovern, S. E., Bakhle, K. M., Hagins, A. C. and Weake, V. M. (2023). Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors. Sci Rep 13(1): 5105. PubMed ID: 36991154
Summary:
Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. This study profiled chromatin marks associated with active transcription in the aging Drosophila eye to understand how chromatin modulates transcriptional outputs. Both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, no correlation was found with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because aging Drosophila undergo a decrease in visual function, these data suggest that H3K36me3 could play a role in maintaining visual function in the aging eye through regulating alternative splicing.
Fellmeth, J. E., Sturm, H., Jang, J., Changela, N., Parikh, A., Persaud, M. and McKim, K. S. (2023). A Dynamic population of prophase CENP-C is required for meiotic chromosome segregation. bioRxiv. PubMed ID: 36993339
Summary:
The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila, which replaces canonical H3 at the centromeres. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. It is not clear, however, if these two functions require the same population of CENP-C. In Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. This study used RNAi knockdown, mutants, and transgenes to study the dynamics and function of CENP-C in meiosis. CENP-C that is incorporated into cells prior to the onset of meiosis is involved in centromere maintenance and CID recruitment. This was not sufficient for the other functions of CENP-C. Indeed, CENP-C is loaded during meiotic prophase, while CID and the chaperone CAL1 are not. CENP-C prophase loading is required for meiotic functions at two different times. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. Thus, CENP-C is one of the few proteins that links the function of the centromeres and kinetochores through the long prophase pause in oocytes.
Terretaz, K., Horard, B., Weill, M., Loppin, B. and Landmann, F. (2023). Functional analysis of Wolbachia Cid effectors unravels cooperative interactions to target host chromatin during replication. PLoS Pathog 19(3): e1011211. PubMed ID: 36928089
Summary:
Wolbachia are common bacteria among terrestrial arthropods. These endosymbionts transmitted through the female germline manipulate their host reproduction through several mechanisms whose most prevalent form called Cytoplasmic Incompatibility -CI- is a conditional sterility syndrome eventually favoring the infected progeny. Upon fertilization, the sperm derived from an infected male is only compatible with an egg harboring a compatible Wolbachia strain, this sperm leading otherwise to embryonic death. The Wolbachia Cif factors CidA and CidB responsible for CI and its neutralization function as a Toxin-Antitoxin system in the mosquito host Culex pipiens. However, the mechanism of CidB toxicity and its neutralization by the CidA antitoxin remain unexplored. Using transfected insect cell lines to perform a structure-function analysis of these effectors, this study shows that both CidA and CidB are chromatin interactors and CidA anchors CidB to the chromatin in a cell-cycle dependent-manner. In absence of CidA, the CidB toxin localizes to its own chromatin microenvironment and acts by preventing S-phase completion, independently of its deubiquitylase -DUB- domain. Experiments with transgenic Drosophila show that CidB DUB domain is required together with CidA during spermatogenesis to stabilize the CidA-CidB complex. This study defines CidB functional regions and paves the way to elucidate the mechanism of its toxicity.
Kochendoerfer, A. M., Keegan, R. S. and Dunleavy, E. M. (2023). Centromere proteins are asymmetrically distributed between newly divided germline stem and daughter cells and maintain a balanced niche in Drosophila males. Mol Biol Cell 34(5): ar42. PubMed ID: 36920070
Summary:
Stem cells can undergo asymmetric cell division (ACD) giving rise to one new stem cell and one differentiating daughter cell. In Drosophila germline stem cells (GSCs), the centromeric histone CENP-A (CID in flies) is asymmetrically distributed between sister chromatids such that chromosomes that end up in the GSC harbor more CID at centromeres. A model of "mitotic drive" has been proposed in GSCs such that stronger and earlier centromere and kinetochore interactions with microtubules bias sister chromatid segregation. This study shows that in Drosophila males, centromere proteins CID, CAL1, and CENP-C are asymmetrically distributed in newly divided GSCs and daughter cells in S phase. Overexpression of CID (either with or without CAL1) or CENP-C depletion disrupts CID asymmetry, with an increased pool of GSCs relative to daughter cells detectable in the niche. This result suggests a shift toward GSC self-renewal rather than differentiation, important for maintaining tissue homeostasis. Overexpression of CAL1 does not disrupt asymmetry, but instead drives germ cell proliferation in the niche. These results in male GSCs are comparable to female GSCs, indicating that despite differences in signaling, organization, and niche composition, the effects of centromere proteins on GSC maintenance are conserved between the sexes.
Sung, H., Vaziri, A., Wilinski, D., Woerner, R. K. R., Freddolino, P. L. and Dus, M. (2023). Nutrigenomic regulation of sensory plasticity. Elife 12. PubMed ID: 36951889
Summary:
Diet profoundly influences brain physiology, but how metabolic information is transmuted into neural activity and behavior changes remains elusive. This study shows that the metabolic enzyme O-GlcNAc Transferase (OGT) moonlights on the chromatin of the D. melanogaster gustatory neurons to instruct changes in chromatin accessibility and transcription that underlie sensory adaptations to a high-sugar diet. OGT works synergistically with the Mitogen Activated Kinase/Extracellular signal Regulated Kinase (MAPK/ERK) rolledand its effector stripe (also known as EGR2 or Krox20) to integrate activity information. OGT also cooperates with the epigenetic silencer Polycomb Repressive Complex 2.1 (PRC2.1) to decrease chromatin accessibility and repress transcription in the high-sugar diet. This integration of nutritional and activity information changes the taste neurons' responses to sugar and the flies' ability to sense sweetness. These findings reveal how nutrigenomic signaling generates neural activity and behavior in response to dietary changes in the sensory neurons.
Ling, L., Muhling, B., Jaenichen, R. and Gompel, N. (2023). Increased chromatin accessibility promotes the evolution of a transcriptional silencer in Drosophila. Sci Adv 9(7): eade6529. PubMed ID: 36800429
Summary:
The loss of discrete morphological traits, the most common evolutionary transition, is typically driven by changes in developmental gene expression. Mutations accumulating in regulatory elements of these genes can disrupt DNA binding sites for transcription factors patterning their spatial expression, or delete entire enhancers. Regulatory elements, however, may be silenced through changes in chromatin accessibility or the emergence of repressive elements. This study shows that increased chromatin accessibility at the gene yellow, combined with the gain of a repressor site, underlies the loss of a wing spot pigmentation pattern in a Drosophila species. The gain of accessibility of this repressive element is regulated by E93, a transcription factor governing the progress of metamorphosis. This convoluted evolutionary scenario contrasts with the parsimonious mutational paths generally envisioned and often documented for morphological losses. It illustrates how evolutionary changes in chromatin accessibility may directly contribute to morphological diversification.

Wednesday, August 2nd - Adult Development

Szlachcic, E., Labecka, A. M., Privalova, V., Sikorska, A. and Czarnoleski, M. (2023). Systemic orchestration of cell size throughout the body: influence of sex and rapamycin exposure in Drosophila melanogaster. Biol Lett 19(3): 20220611. PubMed ID: 36946132
Summary:
Along with differences in life histories, metazoans have also evolved vast differences in cellularity, involving changes in the molecular pathways controlling the cell cycle. The extent to which the signalling network systemically determines cellular composition throughout the body and whether tissue cellularity is organized locally to match tissue-specific functions are unclear. This study cultured genetic lines of Drosophila melanogaster on food with and without rapamycin to manipulate the activity of target of rapamycin (TOR)/insulin pathways and evaluate cell-size changes in five types of adult cells: wing and leg epidermal cells, ommatidial cells, indirect flight muscle cells and Malpighian tubule epithelial cells. Rapamycin blocks TOR multiprotein complex 1, reducing cell growth, but this effect has been studied in single cell types. As adults, rapamycin-treated flies had smaller bodies and consistently smaller cells in all tissues. Regardless, females eclosed with larger bodies and larger cells in all tissues than males. Thus, differences in TOR activity and sex were associated with the orchestration of cell size throughout the body, leading to differences in body size. It is postulated that the activity of TOR/insulin pathways and their effects on cellularity should be considered when investigating the origin of ecological and evolutionary patterns in life histories.
Lim, D. H., Choi, M. S., Jeon, J. W. and Lee, Y. S. (2023). MicroRNA miR-252-5p regulates the Notch signaling pathway by targeting Rab6 in Drosophila wing development. Insect Sci. PubMed ID: 36847222
Summary:
The Notch signaling pathway plays a central role in the development of various organisms. However, dysregulation of microRNAs (miRNAs), which are crucial regulators of gene expression, can disrupt signaling pathways at all stages of development. Although the Notch signaling pathway is involved in wing development in Drosophila, the mechanism underlying miRNA-based regulation of the Notch signaling pathway is unclear. This study reports that loss of Drosophila miR-252 increases the size of adult wings, whereas the overexpression of miR-252 in specific compartments of larval wing discs leads to patterning defects in the adult wings. The miR-252 overexpression-induced wing phenotypes were caused by aberrant Notch signaling with intracellular accumulation of the full-length Notch receptor during development, which could be due to defects in intracellular Notch trafficking associated with its recycling to the plasma membrane and autophagy-mediated degradation. Moreover, Rab6 was identified as a direct target of miR-252-5p; Rab6 encodes a small Ras-like GTPase that regulates endosomal trafficking pathways. Consistent with this finding, RNAi-mediated downregulation of Rab6 led to similar defects in both wing patterning and Notch signaling. Notably, co-overexpression of Rab6 completely rescued the wing phenotype associated with miR-252 overexpression, further supporting that Rab6 is a biologically relevant target of miR-252-5p in the context of wing development. Thus, these data indicate that the miR-252-5p-Rab6 regulatory axis is involved in Drosophila wing development by controlling the Notch signaling pathway.
Rosa, C., Malin, J. and Hatini, V. (2023). Medioapical contractile pulses coordinated between cells regulate Drosophila eye morphogenesis. bioRxiv. PubMed ID: 36993651
Summary:
Lattice cells (LCs) in the developing Drosophila retina constantly move and change shape before attaining final forms. Previous studies showed that repeated contraction and expansion of apical cell contacts affect these dynamics. This study describe sa second contributing factor, the assembly of a medioapical actomyosin ring composed of nodes linked by filaments that attract each other, fuse, and contract the LCs' apical area. This medioapical actomyosin network is dependent on Rho1 and its known effectors. Apical cell area contraction alternates with relaxation, generating pulsatile changes in apical cell area. Strikingly, cycles of contraction and relaxation of cell area are reciprocally synchronized between adjacent LCs. Further, in a genetic screen, RhoGEF2 was identified as an activator of these Rho1 functions and RhoGAP71E/C-GAP as an inhibitor. Thus, Rho1 signaling regulates pulsatile medioapical actomyosin contraction exerting force on neighboring cells, coordinating cell behavior across the epithelium. This ultimately serves to control cell shape and maintain tissue integrity during epithelial morphogenesis of the retina.
Iyer, K. S., Prabhakara, C., Mayor, S. and Rao, M. (2023). Cellular compartmentalisation and receptor promiscuity as a strategy for accurate and robust inference of position during morphogenesis. Elife 12. PubMed ID: 36877545
Summary:
Precise spatial patterning of cell fate during morphogenesis requires accurate inference of cellular position. In making such inferences from morphogen profiles, cells must contend with inherent stochasticity in morphogen production, transport, sensing and signalling. Motivated by the multitude of signalling mechanisms in various developmental contexts, this study shows how cells may utilise multiple tiers of processing (compartmentalisation) and parallel branches (multiple receptor types), together with feedback control, to bring about fidelity in morphogenetic decoding of their positions within a developing tissue. By simultaneously deploying specific and nonspecific receptors, cells achieve a more accurate and robust inference. These ideas were explored in the patterning of Drosophila melanogaster wing imaginal disc by Wingless morphogen signalling, where multiple endocytic pathways participate in decoding the morphogen gradient. The geometry of the inference landscape in the high dimensional space of parameters provides a measure for robustness and delineates stiff and sloppy directions. This distributed information processing at the scale of the cell highlights how local cell autonomous control facilitates global tissue scale design.
Sun, X., Decker, J., Sanchez-Luege, N. and Rebay, I. (2023). Orthogonal coupling of a 3D cytoskeletal scaffold coordinates cell morphogenesis and maintains tissue organization in the Drosophila pupal retina. bioRxiv. PubMed ID: 36945525
Summary:
How complex three-dimensional (3D) organs coordinate cellular morphogenetic events to achieve the correct final form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. This study combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells coordinately organize tissue pattern to support retinal integrity. These experiments revealed an unanticipated intercellular feedback mechanism whereby correct cellular differentiation of either cell type can non-autonomously induce cytoskeletal remodeling in the other Abl mutant cell type, restoring retinal pattern and integrity. It is proposed that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.
Harmansa, S., Erlich, A., Eloy, C., Zurlo, G. and Lecuit, T. (2023). Growth anisotropy of the extracellular matrix shapes a developing organ. Nat Commun 14(1): 1220. PubMed ID: 36869053
Summary:
Final organ size and shape result from volume expansion by growth and shape changes by contractility. Complex morphologies can also arise from differences in growth rate between tissues. This study addresses how differential growth guides the morphogenesis of the growing Drosophila wing imaginal disc. 3D morphology results from elastic deformation due to differential growth anisotropy between the epithelial cell layer and its enveloping extracellular matrix (ECM). While the tissue layer grows in plane, growth of the bottom ECM occurs in 3D and is reduced in magnitude, thereby causing geometric frustration and tissue bending. The elasticity, growth anisotropy and morphogenesis of the organ are fully captured by a mechanical bilayer model. Moreover, differential expression of the Matrix metalloproteinase MMP2 controls growth anisotropy of the ECM envelope. This study shows that the ECM is a controllable mechanical constraint whose intrinsic growth anisotropy directs tissue morphogenesis in a developing organ.

Tuesday, August 1st - Physiology and Metabolism

Kotronarou, K., Charalambous, A., Evangelou, A., Georgiou, O., Demetriou, A. and Apidianakis, Y. (2023). Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate. Metabolites 13(2). PubMed ID: 36837883
Summary:
Peptide hormones control Drosophila gut motility, but the intestinal stimuli and the gene networks coordinating this trait remain poorly defined. This study customized an assay to quantify female Drosophila defecation rate as a proxy of intestinal motility. Bacterial infection with the human opportunistic bacterial pathogen Pseudomonas aeruginosa (strain PA14) was found to increase defecation rate in wild-type female flies, and specific bacteria of the fly microbiota able to increase defecation rate were identified. In contrast, dietary stress, imposed by either water-only feeding or high ethanol consumption, decreased defecation rate and the expression of enteroendocrine-produced hormones in the fly midgut, such as Diuretic hormone 31 (Dh31). The decrease in defecation due to dietary stress was proportional to the impact of each stressor on fly survival. Furthermore, the Drosophila Genetic Reference Panel wild type strain collection was exploited, and strains displaying high and low defecation rates were identifed. The narrow-sense heritability of defecation rate was calculated to be 91%, indicating that the genetic variance observed using the current assay is mostly additive and polygenic in nature. Accordingly, a genome-wide association (GWA) analysis was performed revealing 17 candidate genes linked to defecation rate. Downregulation of four of them (Pmp70, CG11307, meso18E and mub) in either the midgut enteroendocrine cells or in neurons reduced defecation rate and altered the midgut expression of Dh31, that in turn regulates defecation rate via signaling to the visceral muscle. Hence, microbial and dietary stimuli, and Dh31-controlling genes, regulate defecation rate involving signaling within and among neuronal, enteroendocrine, and visceral muscle cells.
Horvath, V., Guirao-Rico, S., Salces-Ortiz, J., Rech, G. E., Green, L., Aprea, E., Rodeghiero, M., Anfora, G. and Gonzalez, J. (2023). Gene expression differences consistent with water loss reduction underlie desiccation tolerance of natural Drosophila populations. BMC Biol 21(1): 35. PubMed ID: 36797754
Summary:
Climate change is one of the main factors shaping the distribution and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. However, most of the studies so far have focused on the effects of increased temperature. Integrating transcriptomics and physiology is key to advancing knowledge on how species cope with desiccation stress, and these studies are still best accomplished in model organisms. This study characterized the natural variation of European D. melanogaster populations across climate zones and found that strains from arid regions were similar or more tolerant to desiccation compared with strains from temperate regions. Tolerant and sensitive strains differed not only in their transcriptomic response to stress but also in their basal expression levels. It was further shown that gene expression changes in tolerant strains correlated with their physiological response to desiccation stress and with their cuticular hydrocarbon composition, and functionally validated three of the candidate genes identified. Transposable elements, which are known to influence stress response across organisms, were not found to be enriched nearby differentially expressed genes. Finally, several tRNA-derived small RNA fragments were identified that differentially targeted genes in response to desiccation stress. Overall, these results showed that basal gene expression differences across individuals should be analyzed if the genetic basis of differential stress survival is to be understood. Moreover, tRNA-derived small RNA fragments appear to be relevant across stress responses and allow for the identification of stress-response genes not detected at the transcriptional level.
Haga-Yamanaka, S., Nunez-Flores, R., Scott, C. A., Perry, S., Chen, S. T., Pontrello, C., Nair, M. G. and Ray, A. (2023). Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs. bioRxiv. PubMed ID: 36865229
Summary:
Eukaryotes are often exposed to microbes and respond to their secreted metabolites, such as the microbiome in animals or commensal bacteria in roots. Little is known about the effects of long-term exposure to volatile chemicals emitted by microbes, or other volatiles that humans are exposed to over a long duration. Using the model system Drosophila melanogaster this study evaluated a yeast emitted volatile, diacetyl, found in high levels around fermenting fruits where they spend long periods of time. Exposure to just the headspace containing the volatile molecules was shown to alter gene expression in the antenna. Experiments showed that diacetyl and structurally related volatile compounds inhibited human histone-deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused wide changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure causes modulation of gene expression in the brain, therefore has potential as a therapeutic. Using two separate disease models known to be responsive to HDAC-inhibitors, this study evaluated physiological effects of volatile exposure. First, it was found that the HDAC inhibitor also halts proliferation of a neuroblastoma cell line in culture as predicted. Next, exposure to vapors slows progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression and physiology in animals.
Jang, W., Oh, M., Cho, E. H., Baek, M. and Kim, C. (2023).. Drosophila pain sensitization and modulation unveiled by a novel pain model and analgesic drugs. PLoS One 18(2): e0281874. PubMed ID: 36795675
Summary:
In mammals, pain is regulated by the combination of an ascending stimulating and descending inhibitory pain pathway. It remains an intriguing question whether such pain pathways are of ancient origin and conserved in invertebrates. This study reports a new Drosophila pain model and use it to elucidate the pain pathways present in flies. The model employs transgenic flies expressing the human capsaicin receptor TRPV1 in sensory nociceptor neurons, which innervate the whole fly body, including the mouth. Upon capsaicin sipping, the flies abruptly displayed pain-related behaviors such as running away, scurrying around, rubbing vigorously, and pulling at their mouth parts, suggesting that capsaicin stimulated nociceptors in the mouth via activating TRPV1. When reared on capsaicin-containing food, the animals died of starvation, demonstrating the degree of pain experienced. This death rate was reduced by treatment both with NSAIDs and gabapentin, analgesics that inhibit the sensitized ascending pain pathway, and with antidepressants, GABAergic agonists, and morphine, analgesics that strengthen the descending inhibitory pathway. These results suggest Drosophila to possess intricate pain sensitization and modulation mechanisms similar to mammals, and it is proposed that this simple, non-invasive feeding assay has utility for high-throughput evaluation and screening of analgesic compounds.
Jneid, R., Loudhaief, R., Zucchini-Pascal, N., Nawrot-Esposito, M. P., Fichant, A., Rousset, R., Bonis, M., Osman, D. and Gallet, A. (2023). Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila. Elife 12. PubMed ID: 36847614
Summary:
Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. This study shows that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.
Lee, H. Y., Lee, B., Lee, E. J. and Min, K. J. (2023). Effects of Parental Dietary Restriction on Offspring Fitness in Drosophila melanogaster. Nutrients 15(5). PubMed ID: 36904272
Summary:
Dietary restriction (DR) is a well-established strategy to increase lifespan and stress resistance in many eukaryotic species. In addition, individuals fed a restricted diet typically reduce or completely shut down reproduction compared to individuals fed a full diet. Although the parental environment can lead to changes epigenetically in offspring gene expression, little is known about the role of the parental (F(0)) diet on the fitness of their offspring (F(1)). This study investigated the lifespan, stress resistance, development, body weight, fecundity, and feeding rate in offspring from parental flies exposed to a full or restricted diet. The offspring flies of the parental DR showed increases in body weight, resistance to various stressors, and lifespan, but the development and fecundity were unaffected. Interestingly, parental DR reduced the feeding rate of their offspring. This study suggests that the effect of DR can extend beyond the exposed individual to their offspring, and it should be considered in both theoretical and empirical studies of senescence. Home page: The Interactive Fly© 2020 Thomas B. Brody, Ph.D.

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