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Tuesday, November 29th, 2022 - Behavior

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December 2019 		Fowler, E. K., Leigh, S., Bretman, A. and Chapman, T. (2022). Plastic responses of males and females interact to determine mating behavior. Evolution 76(9): 2116-2129. PubMed ID: 35880536
Summary:
Individuals can respond plastically to variation in their social environment. However, each sex may respond to different cues and contrasting aspects of competition. Theory suggests that the plastic phenotype expressed by one sex can influence evolutionary dynamics in the other, and that plasticity simultaneously expressed by both sexes can exert sex-specific effects on fitness. However, data are needed to test this theory base. This study examined whether the simultaneous expression of adaptive plasticity by both sexes of Drosophila melanogaster fruit flies in response to their respective social environments interacts to determine the value of key reproductive traits (mating latency, duration, and fecundity). To vary social environments, males were kept alone, or with same sex rivals, and females were kept alone, in same-sex, or mixed-sex groups. Matings were then conducted between individuals from all of these five social treatments in all combinations, and the resulting reproductive traits measured in both "choice" and "no-choice" assays. Mating latency was determined by an interaction between the plastic responses of both sexes to their social environments. Interestingly, the mating latency response occurred in opposing directions in the different assays. In females exposed to same-sex social treatments, mating latency was more rapid with rival treatment males in the choice assays, but slower with those same males in no-choice assays. In contrast, mating duration was determined purely by responses of males to their social environments, and fecundity purely by responses of females. Collectively, the results show that plastic responses represent an important and novel facet of sexual interactions. 		Au, D. D., Foden, A. J., Park, S. J., Nguyen, T. H., Liu, J. C., Tran, M. D., Jaime, O. G., Yu, Z. and Holmes, T. C. (2022). Mosquito cryptochromes expressed in Drosophila confer species-specific behavioral light responses. Curr Biol 32(17): 3731-3744. PubMed ID: 35914532
Summary:
Cryptochrome (CRY) is a short-wavelength light-sensitive photoreceptor expressed in a subset of circadian neurons and eyes in Drosophila that regulates light-evoked circadian clock resetting. Acutely, light evokes rapid electrical excitation of the ventral lateral subset of circadian neurons and confers circadian-modulated avoidance behavioral responses to short-wavelength light. Recent work shows dramatically different avoidance versus attraction behavioral responses to short-wavelength light in day-active versus night-active mosquitoes. To determine whether CRY1s mediate species-specific coding for behavioral and electrophysiological light responses,an "empty neuron" approach was used and transgenically expressed diurnal Aedes aegypti (AeCRY1) versus nocturnal Anopheles gambiae (AgCRY1) in a cry-null Drosophila background. AeCRY1 is much less light sensitive than either AgCRY1 or DmCRY as shown by partial behavioral rhythmicity following constant light exposure. Remarkably, expression of nocturnal AgCRY1 confers low survival to constant white light as does expression of AeCRY1 to a lesser extent. AgCRY1 mediates significantly stronger electrophysiological cell-autonomous responses to 365 nm ultraviolet (UV) light relative to AeCRY1. AgCRY1 expression mediates electrophysiological sensitivity to 635 nm red light, whereas AeCRY1 does not, consistent with species-specific mosquito red light responses. AgCRY1 and DmCRY mediate intensity-dependent avoidance behavior to UV light at different light intensity thresholds, whereas AeCRY1 does not, thus mimicking mosquito and fly behaviors. These findings highlight CRY as a key non-image-forming visual photoreceptor that mediates physiological and behavioral light responses in a species-specific fashion.
Gaspar, M., Dias, S. and Vasconcelos, M. L. (2022). Mating pair drives aggressive behavior in female Drosophila. Curr Biol 32(21): 4734-4742. PubMed ID: 36167074
Summary:
Aggression is an adaptive set of behaviors that allows animals to compete against one another in an environment of limited resources. Typically, males fight for mates and food, whereas females fight for food and nest sites. Although the study of male aggression has been facilitated by the extravagant nature of the ritualized displays involved and the remarkable armaments sported by males of many species the subtler and rarer instances of inter-female aggression have historically received much less attention. In Drosophila, females display high levels of complex and highly structured aggression on a food patch with conspecific females. Other contexts of female aggression have not been explored. Indeed, whether females compete for mating partners, as males do, has remained unknown so far. In the present work, it is reported that Drosophila melanogaster females reliably display aggression toward mating pairs. This aggressive behavior is regulated by mating status and perception of mating opportunities and relies heavily on olfaction. Furthermore, food odor in combination with OR47b-dependent fly odor sensing is required for proper expression of aggressive behavior. Taken together, this study describes a social context linked to reproduction in which Drosophila females aspiring to mate produce consistent and stereotyped displays of aggression. These findings open the door for further inquiries into the neural mechanisms that govern this behavior. 		Fanara, J. J., Beti, M. I. L., Gandini, L. and Hasson, E. (2022). Oviposition behaviour in Drosophila melanogaster: Genetic and behavioural decoupling between oviposition acceptance and preference for natural fruits. J Evol Biol. PubMed ID: 36357966
Summary:
In phytophagous insects, oviposition behaviour is an important component of habitat selection and, given the multiplicity of genetic and environmental factors affecting its expression, is defined as a complex character resulting from the sum of interdependent traits. This study examined two components of egg-laying behaviour: oviposition acceptance (OA) and oviposition preference (OP) in Drosophila melanogaster using three natural fruits as resources (grape, tomato and orange) by means of no-choice and two-choice experiments, respectively. This experimental design showed that the results obtained in two-choice assays (OP) cannot be accounted for by those resulting from no-choice assays (OA). Since the genomes of all lines used are completely sequenced, a genome-wide association study was performed to identify and characterize the genetic underpinnings of these oviposition behaviour traits. The analyses revealed different candidate genes affecting natural genetic variation of both OA and OP traits. Moreover, the results suggest behavioural and genetic decoupling between OA and OP and that egg-laying behaviour is plastic and context-dependent. Such independence in the genetic architectures of OA and OP variation may influence different aspects of oviposition behaviour, including plasticity, canalization, host shift and maintenance of genetic variability, which contributes to the adoption of adaptive strategies during habitat selection.
Das, D., Begum, M., Paul, P., Dutta, I., Mandal, S., Ghosh, P. and Ghosh, S. (2022). Effects of plant growth retardant daminozide (Alar) on neuromuscular co-ordination behavior in Drosophila melanogaster. J Toxicol Environ Health A 85(22): 921-936. PubMed ID: 35996764
Summary:
Daminozide (alar), a plant growth retardant, is used in different fruit orchard to make fruits attractive and reduce pre-harvest losses. Previously data demonstrated that acute daminozide exposure affected reproductive fitness and produced neurodegeneration in Drosophila melanogaster. The goal of this study was to determine whether continuous exposure to daminozide affects neuromuscular co-ordination in D. melanogaster as manifested in various behavioral responses. Fruit flies were exposed to 200 or 400 mg/L concentration of daminozide for two successive generations. Treated D. melanogaster were examined for the behaviors indicative of neuromuscular coordination and cognitive abilities, that include climbing, social interaction, adult grooming, migration, flight, male aggression, and adult courtship. Aberrant behavioral responses were noted among treated D. melanogaster of both sexes as evidenced by the following parameters: reduction in flight duration, abnormal social interaction, altered copulatory acts, and over-aggressiveness. Data suggest that daminozide produces impairment in neuromuscular coordination and cognitive ability in Drosophila, which was reflected as altered behavioral patterns. As Drosophila is considered as a reliable in vivo model utilized in toxicity testing, our findings may help us to anticipate and monitor potential daminozide-induced toxicity in animals and humans. 		Tang, M., Cao, L. H., Yang, T., Ma, S. X., Jing, B. Y., Xiao, N., Xu, S., Leng, K. R., Yang, D., Li, M. T. and Luo, D. G. (2022). An extra-clock ultradian brain oscillator sustains circadian timekeeping. Sci Adv 8(35): eabo5506. PubMed ID: 36054358
Summary:
The master circadian clock generates 24-hour rhythms to orchestrate daily behavior, even running freely under constant conditions. Traditionally, the master clock is considered self-sufficient in sustaining free-running timekeeping via its cell-autonomous molecular clocks and interneuronal communications within the circadian neural network. This study found a set of bona fide ultradian oscillators in the Drosophila brain that support free-running timekeeping, despite being located outside the master clock circuit and lacking clock gene expression. These extra-clock electrical oscillators (xCEOs) generate cell-autonomous ultradian bursts, pacing widespread burst firing and promoting rhythmic resting membrane potentials in clock neurons via parallel monosynaptic connections. Silencing xCEOs disrupts daily electrical rhythms in clock neurons and impairs cycling of neuropeptide pigment dispersing factor, leading to the loss of free-running locomotor rhythms. Together, it is concluded that the master clock is not self-sufficient to sustain free-running behavior rhythms but requires additional endogenous inputs to the clock from the extra-clock ultradian brain oscillators.

Monday, November 28th - Larval and Adult Neural Development and Function

Zheng, Z., Li, F., Fisher, C., Ali, I. J., Sharifi, N., Calle-Schuler, S., Hsu, J., Masoodpanah, N., Kmecova, L., Kazimiers, T., Perlman, E., Nichols, M., Li, P. H., Jain, V. and Bock, D. D. (2022). Structured sampling of olfactory input by the fly mushroom body. Curr Biol 32(15): 3334-3349. PubMed ID: 35797998
Summary:
Associative memory formation and recall in the fruit fly Drosophila melanogaster is subserved by the mushroom body (MB). Upon arrival in the MB, sensory information undergoes a profound transformation from broadly tuned and stereotyped odorant responses in the olfactory projection neuron (PN) layer to narrowly tuned and nonstereotyped responses in the Kenyon cells (KCs). Theory and experiment suggest that this transformation is implemented by random connectivity between KCs and PNs. However, this hypothesis has been challenging to test, given the difficulty of mapping synaptic connections between large numbers of brain-spanning neurons. This study used a recent whole-brain electron microscopy volume of the adult fruit fly to map PN-to-KC connectivity at synaptic resolution. The PN-KC connectome revealed unexpected structure, with preponderantly food-responsive PN types converging at above-chance levels on downstream KCs. Axons of the overconvergent PN types tended to arborize near one another in the MB main calyx, making local KC dendrites more likely to receive input from those types. Overconvergent PN types preferentially co-arborize and connect with dendrites of αβ and α'β' KC subtypes. Computational simulation of the observed network showed degraded discrimination performance compared with a random network, except when all signal flowed through the overconvergent, primarily food-responsive PN types. Additional theory and experiment will be needed to fully characterize the impact of the observed non-random network structure on associative memory formation and recall. 		Cho, J. H., Jo, M. G., Kim, E. S., Lee, N. Y., Kim, S. H., Chung, C. G., Park, J. H. and Lee, S. B. (2022). CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons. Mol Cells 45(11): 855-867. PubMed ID: 36172977
Summary:
For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, this study investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, dynamic changes were observed in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. It was further shown that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.
Dzaki, N., Bu, S., Lau, S. S. Y., Yong, W. L. and Yu, F. (2022). Drosophila GSK3β promotes microtubule disassembly and dendrite pruning in sensory neurons. Development 149(22). PubMed ID: 36264221
Summary:
The evolutionarily conserved Glycogen Synthase Kinase 3β (GSK3β), a negative regulator of microtubules, is crucial for neuronal polarization, growth and migration during animal development. However, it remains unknown whether GSK3β regulates neuronal pruning, which is a regressive process. This study reports that the Drosophila GSK3β homologue Shaggy (Sgg) is cell-autonomously required for dendrite pruning of ddaC sensory neurons during metamorphosis. Sgg is necessary and sufficient to promote microtubule depolymerization, turnover and disassembly in the dendrites. Although Sgg is not required for the minus-end-out microtubule orientation in dendrites, hyperactivated Sgg can disturb the dendritic microtubule orientation. Moreover, pharmacological and genetic data suggest that Sgg is required to promote dendrite pruning at least partly via microtubule disassembly. We show that Sgg and Par-1 kinases act synergistically to promote microtubule disassembly and dendrite pruning. Thus, Sgg and Par-1 might converge on and phosphorylate a common downstream microtubule-associated protein(s) to disassemble microtubules and thereby facilitate dendrite pruning. 		Vaughen, J. P., Theisen, E., Rivas-Serna, I. M., Berger, A. B., Kalakuntla, P., Anreiter, I., Mazurak, V. C., Rodriguez, T. P., Mast, J. D., Hartl, T., Perlstein, E. O., Reimer, R. J., Clandinin, M. T. and Clandinin, T. R. (2022). Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit. Neuron. PubMed ID: 35961319
Summary:
Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. This study shows that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.
Thoener, J., Weiglein, A., Gerber, B. and Schleyer, M. (2022). Optogenetically induced reward and 'frustration' memory in larval Drosophila melanogaster. J Exp Biol 225(16). PubMed ID: 35924545
Summary:
Animals, including humans, form oppositely valenced memories for stimuli that predict the occurrence versus the termination of a reward: appetitive 'reward' memory for stimuli associated with the occurrence of a reward and aversive 'frustration' memory for stimuli that are associated with its termination. This study characterized these memories in larval Drosophila melanogaster using a combination of Pavlovian conditioning, optogenetic activation of the dopaminergic central-brain DAN-i1864 neuron, and high-resolution video-tracking. This reveals their dependency on the number of training trials and the duration of DAN-i1864 activation, their temporal stability, and the parameters of locomotion that are modulated during memory expression. Together with previous results on 'punishment' versus 'relief' learning by DAN-f1 neuron activation, this reveals a 2×2 matrix of timing-dependent memory valence for the occurrence/termination of reward/punishment. These findings should aid the understanding and modelling of how brains decipher the predictive, causal structure of events around a target reinforcing occurrence. 		Corty, M. M., Hulegaard, A. L., Hill, J. Q., Sheehan, A. E., Aicher, S. A. and Freeman, M. R. (2022). Discoidin domain receptor regulates ensheathment, survival, and caliber of peripheral axons. Development. PubMed ID: 36355066
Summary:
Most invertebrate axons and small caliber axons in mammalian peripheral nerves are unmyelinated but still ensheathed by glia. This study used Drosophila wrapping glia to study the development and function of non-myelinating axon ensheathment, which is poorly understood. Selective ablation of these glia from peripheral nerves severely impaired larval locomotor behavior. In an in vivo RNAi screen to identify glial genes required for axon ensheathment, the conserved receptor tyrosine kinase Discoidin domain receptor (Ddr) was identified. In larval peripheral nerves, loss of Ddr resulted in severely reduced ensheathment of axons and reduced axon caliber, and a strong dominant genetic interaction was found between Ddr and the type XV/XVIII collagen Multiplexin (Mp), suggesting Ddr functions as a collagen receptor to drive axon wrapping. In adult nerves, loss of Ddr decreased long-term survival of sensory neurons and significantly reduced axon caliber without overtly affecting ensheathment. These data establish essential roles for non-myelinating glia in nerve development, maintenance, and function, and identify Ddr as a key regulator of axon-glia interactions during ensheathment and establishment of axon caliber.

Friday, November 25th - Disease Models

Ataellahi, F., Masoudi, R. and Haddadi, M. (2022). Differential dysregulation of CREB and synaptic genes in transgenic Drosophila melanogaster expressing shaggy (GSK3), Tau(WT), or Amyloid-beta. Mol Biol Rep. PubMed ID: 36399243
Summary:
Tau, Amyloid-beta (Aβ42), and Glycogen synthase kinase 3 (GSK3) contribute to synaptic dysfunction observed in Alzheimer's disease (AD), the most common form of dementia. In the current study, the effect of pan-neuronal expression of TauWT, Aβ42, or shaggy (orthologue of GSK3) in Drosophila melanogaster was assessed on the locomotor function, ethanol sensitivity, synaptic genes and CREB expression. The effect of TauWT and Aβ42 on the expression of shaggy was also determined. Gene expression analysis was performed using quantitative real-time RT-PCR method. While syt1, SNAP25 and CREB (upstream transcription factor of syt1 and SNAP25) were upregulated in flies expressing Tau(WT) or Aβ42, a prominent decline was observed in those genes in shaggy expressing flies. Although all transgenic flies showed climbing disability and higher sensitivity to ethanol, abnormality in these features was significantly more prominent in transgenic flies expressing shaggy compared to TauWT or Aβ42. Despite a significant upregulation of shaggy transcription in TauWT expressing flies, Aβ42 transgenic flies witnessed no significant changes. TauWT, A&beta42, and shaggy may affect synaptic plasticity through dysregulation of synaptic genes and CREB, independently. However shaggy has more detrimental effect on synaptic genes expression, locomotor ability and sensitivity to ethanol. It is important when it comes to drug discovery. It appears that CREB is a direct effector of changes in synaptic genes expression as they showed similar pattern of alteration and it is likely to be a part of compensatory mechanisms independent of the GSK3/CREB pathway in TauWT or Aβ(42) expressing flies. 		Ozaki, M., Le, T. D. and Inoue, Y. H. (2022). Downregulating Mitochondrial DNA Polymerase γ in the Muscle Stimulated Autophagy, Apoptosis, and Muscle Aging-Related Phenotypes in Drosophila Adults. Biomolecules 12(8). PubMed ID: 36008999
Summary:
Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. This study investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. Our findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging.
Wang, R., Zhang, P., Wang, J., Ma, L., E, W., Suo, S., Jiang, M., Li, J., Chen, H., Sun, H., Fei, L., Zhou, Z., Zhou, Y., Chen, Y., Zhang, W., Wang, X., Mei, Y., Sun, Z., Yu, C., Shao, J., Fu, Y., Xiao, Y., Ye, F., Fang, X., Wu, H., Guo, Q., Fang, X., Li, X., Gao, X., Wang, D., Xu, P. F., Zeng, R., Xu, G., Zhu, L., Wang, L., Qu, J., Zhang, D., Ouyang, H., Huang, H., Chen, M., Ng, S. C., Liu, G. H., Yuan, G. C., Guo, G. and Han, X. (2022). Construction of a cross-species cell landscape at single-cell level. Nucleic Acids Res. PubMed ID: 35929025
Summary:
w Individual cells are basic units of life. Despite extensive efforts to characterize the cellular heterogeneity of different organisms, cross-species comparisons of landscape dynamics have not been achieved. This study applied single-cell RNA sequencing (scRNA-seq) to map organism-level cell landscapes at multiple life stages for mice, zebrafish and Drosophila. By integrating the comprehensive dataset of > 2.6 million single cells, a cross-species cell landscape was developed, and signatures and common pathways were identified that changed throughout the life span. structural inflammation and mitochondrial dysfunction were identified as the most common hallmarks of organism aging, and pharmacological activation of mitochondrial metabolism was found to alleviated aging phenotypes in mice. The cross-species cell landscape with other published datasets were stored in an integrated online portal-Cell Landscape. This work provides a valuable resource for studying lineage development, maturation and aging. How many cell types are there in nature? How do they change during the life cycle? These are two fundamental questions that researchers have been trying to understand in the area of biology. In this study, single-cell mRNA sequencing data were used to profile over 2.6 million individual cells from mice, zebrafish and Drosophila at different life stages, 1.3 million of which were newly collected. The comprehensive datasets allow investigators to construct a cross-species cell landscape that helps to reveal the conservation and diversity of cell taxonomies at genetic and regulatory levels. The resources in this study are assembled into a publicly available at website 		Riera-Escamilla, A., Vockel, M., Nagirnaja, L., Xavier, M. J., Carbonell, A., Moreno-Mendoza, D., Pybus, M., Farnetani, G., Rosta, V., Cioppi, F., Friedrich, C., Oud, M. S., van der Heijden, G. W., Soave, A., Diemer, T., Ars, E., Sanchez-Curbelo, J., Kliesch, S., O'Bryan, M. K., Ruiz-Castañe, E., Azorín, F., Veltman, J. A., Aston, K. I., Conrad, D. F., Tuttelmann, F. and Krausz, C. (2022). Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure. Am J Hum Genet 109(8): 1458-1471. PubMed ID: 35809576
Summary:
Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. This study aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, it is proposed that 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, this study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.
Choi, B., Lim, C., Lee, H., Lee, J. E., Kim, J., Chung, C. and Cho, K. S. (2022). Neuroprotective effects of linear ubiquitin E3 ligase against aging-induced DNA damage and amyloid β neurotoxicity in the brain of Drosophila melanogaster. Biochem Biophys Res Commun 637: 196-202. PubMed ID: 36403483
Summary:
E3 ubiquitin ligase, HOIL1-interacting protein (HOIP), forms the linear ubiquitin chain assembly complex (LUBAC) with HOIL and SHANK-associated RH domain interactor and catalyzes linear ubiquitination, directly linking the N- and C-termini of ubiquitin. Recently, several studies have implicated linear ubiquitination in aging and Alzheimer disease (AD). However, little is currently known about the roles of HOIP in brain aging and AD pathology. This studyinvestigated the role of linear ubiquitin E3 ligase (LUBEL), a Drosophila HOIP ortholog, in brain aging and amyloid β (A:beta;) pathology in a Drosophila AD model. DNA double-strand breaks (DSBs) were increased in the aged brains of neuron-specific LUBEL-knockdown flies compared to the age-matched controls. Silencing of LUBEL in the neuron of AD model flies increased the neuronal apoptosis and neurodegeneration, whereas silencing in glial cells had no such effect. A&bet; aggregation levels and DSBs were also increased in the LUBEL-silenced AD model fly brains, but autophagy and proteostasis were not affected by LUBEL silencing. Collectively, these results suggest that LUBEL protects neurons from aging-induced DNA damage and Aβ neurotoxicity. 		Chen, Y., Krishnan, G., Parsi, S., Pons, M., Nikolaki, V., Cao, L., Xu, Z. and Gao, F. B. (2022). The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias. Acta Neuropathol Commun 10(1): 169. PubMed ID: 36414997
Summary:
Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2B(Intron5). Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. This study found that Spastin binds with greater affinity to CHMP2B(Intron5) than to CHMP2B(WT) and colocalizes with CHMP2B(Intron5) in p62-positive aggregates. In cultured cells expressing CHMP2B(Intron5), spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2B(Intron5) toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, this study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Wednesday, November 23 - Signal Transduction

Savola, E., Vale, P. F. and Walling, C. A. (2022). Larval diet affects adult reproduction, but not survival, independent of the effect of injury and infection in Drosophila melanogaster. J Insect Physiol 142: 104428. PubMed ID: 35932926
Summary:
Early-life conditions have profound effects on many life-history traits, where early-life diet affects both juvenile development, and adult survival and reproduction. Early-life diet also has consequences for the ability of adults to withstand environmental challenges such as starvation, temperature and desiccation. However, it is less well known how early-life diet influences the consequences of infection in adults. This study tested whether varying the larval diet of female Drosophila melanogaster (through altering protein to carbohydrate ratio, P:C) influences the long-term consequences of injury and infection with the bacterial pathogen Pseudomonasentomophila. Given previous work manipulating adult dietary P:C, it was predicted that adults from larvae raised on higher P:C diets would have increased reproduction, but shorter lifespans and an increased rate of ageing, and that the lowest larval P:C diets would be particularly detrimental for adult survival in infected individuals. For larval development, it was predicted that low P:C would lead to a longer development time and lower viability. Early-life and lifetime egg production were highest at intermediate to high larval P:C diets, but this was independent of injury and infection. There was no effect of larval P:C on adult survival. Larval development was quickest on intermediate P:C and egg-to-pupae and egg-to-adult viability were slightly higher on higher P:C. Overall, despite larval P:C affecting several measured traits, no evidence was seen that larval P:C altered the consequence of infection or injury for adult survival or early-life and lifetime reproduction. Taken together, these data suggest that larval diets appear to have a limited impact on the adult life history consequences of infection. 		Zhou, S., Lu, Y., Chen, J., Pan, Z., Pang, L., Wang, Y., Zhang, Q., Strand, M. R., Chen, X. X. and Huang, J. (2022). Parasite reliance on its host gut microbiota for nutrition and survival. Isme j. PubMed ID: 35941172
Summary:
Studying the microbial symbionts of eukaryotic hosts has revealed a range of interactions that benefit host biology. Most eukaryotes are also infected by parasites that adversely affect host biology for their own benefit. However, it is largely unclear whether the ability of parasites to develop in hosts also depends on host-associated symbionts, e.g., the gut microbiota. The effects were studied of parasitic wasp Leptopilina boulardi (Lb) and its host Drosophila melanogaster. Results showed that Lb successfully develops in conventional hosts (CN) with a gut microbiota but fails to develop in axenic hosts (AX) without a gut microbiota. Developing Lb larvae consume fat body cells that store lipids. It was also determined that much larger amounts of lipid accumulate in fat body cells of parasitized CN hosts than parasitized AX hosts. CN hosts parasitized by Lb exhibited large increases in the abundance of the bacterium Acetobacter pomorum in the gut, but did not affect the abundance of Lactobacillus fructivorans which is another common member of the host gut microbiota. However, AX hosts inoculated with A. pomorum and/or L. fructivorans did not rescue development of Lb. In contrast, AX larvae inoculated with A. pomorum plus other identified gut community members including a Bacillus sp. substantially rescued Lb development. Rescue was further associated with increased lipid accumulation in host fat body cells. Insulin-like peptides increased in brain neurosecretory cells of parasitized CN larvae. Lipid accumulation in the fat body of CN hosts was further associated with reduced Bmm lipase activity mediated by insulin/insulin-like growth factor signaling (IIS). Altogether, these results identify a previously unknown role for the gut microbiota in defining host permissiveness for a parasite. These findings also identify a new paradigm for parasite manipulation of host metabolism that depends on insulin signaling and the gut microbiota.
Kumar, M., Has, C., Lam-Kamath, K., Ayciriex, S., Dewett, D., Bashir, M., Poupault, C., Schuhmann, K., Knittelfelder, O., Raghuraman, B. K., Ahrends, R., Rister, J. and Shevchenko, A. (2022). Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Drosophila Eye Proteome. Biomolecules 12(8). PubMed ID: 36008977
Summary:
The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, this study took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. The impacts were analyzed of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. Chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, no apparent effects of vitamin A deficiency on the ocular lipidome were found. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated. 		Lee, S. A., Kim, V., Choi, B., Lee, H., Chun, Y. J., Cho, K. S. and Kim, D. (2022). Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase. Biomol Ther (Seoul). PubMed ID: 35934685
Summary:
Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. This study charaterized he biochemical properties of CYP6A8. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni(2+)-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with K(d) values 28 ± 4 and 144 ± 20 μM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and &ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k(cat) value of 0.038 ± 0.002 min(-1) and a K(m) value of 10 ± 2 μM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k(cat) value of 0.135 ± 0.007 min(-1) and a K(m) value of 21 ± 4 M. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.
Zhong, L., Tang, H., Xu, Y., Liu, X., Shan, J. and Shen, J. (2022). Luteolin alleviated damage caused by blue light to Drosophila. Photochem Photobiol Sci. PubMed ID: 35930192
Summary:
Short-wavelength blue light is commonly found in daily life and is harmful to health. In this experiment, the effect of luteolin was investigated on the survival time of Drosophila under the blue light condition of 3000 Lux using Drosophila as the model organism. The results showed that luteolin alleviated the damage suffered by Drosophila under blue light irradiation, significantly prolonged the survival time of Drosophila, prolonged the survival time of male Drosophila in the heat stress assay, increased the activity of female Drosophila in the spontaneous activity assay, and increased the egg production of female Drosophila at the highest concentration, and there was no significant difference in the food intake experiment. It is suggested that the increase in survival time of Drosophila under blue light conditions is due to the function of luteolin in resisting oxidative stress. 		Shi, K. and Tong, C. (2022). Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body. J Vis Exp(186). PubMed ID: 35993761
Summary:
Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans. The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Monday, September 21 - Cytoskeleton and Junctions

Zhao, A. J., Montes-Laing, J., Perry, W. M. G., Shiratori, M., Merfeld, E., Rogers, S. L. and Applewhite, D. A. (2022). The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin. Mol Biol Cell 33(5): ar19. PubMed ID: 35235367
Summary:
The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. This study examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted an analysis of focal adhesion dynamics. The rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, it was found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion. 		Henry, S. M., Xie, Y., Rollins, K. R. and Blankenship, J. T. (2022). Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression. Dev Biol 491: 82-93. PubMed ID: 36067836
Summary:
In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10-13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. In this study quantitative analysis was performed of the effects of Sponge disruption on cortical cap growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. Membrane trafficking is relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but that Sponge acts as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, it is also suggested that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, the phasic requirements for branched F-actin and linear F-actin networks in potentiating furrow ingression was demonstrated. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows.
Xiang, W., Zur Lage, P., Newton, F. G., Qiu, G. and Jarman, A. P. (2022). The dynamics of protein localisation to restricted zones within Drosophila mechanosensory cilia. Sci Rep 12(1): 13338. PubMed ID: 35922464
Summary:
The Drosophila chordotonal neuron cilium is the site of mechanosensory transduction. The cilium has a 9 + 0 axoneme structure and is highly sub-compartmentalised, with proximal and distal zones harbouring different TRP channels and the proximal zone axoneme also being decorated with axonemal dynein motor complexes. The activity of the dynein complexes is essential for mechanotransduction. The localisation of TRP channels and dynein motor complexes during ciliogenesis was investigated. Differences in timing of TRP channel localisation correlate with order of construction of the two ciliary zones. Dynein motor complexes are initially not confined to their target proximal zone, but ectopic complexes beyond the proximal zone are later cleared, perhaps by retrograde transport. Differences in transient distal localisation of outer and inner dynein arm complexes (ODAs and IDAs) are consistent with previous suggestions from unicellular eukaryotes of differences in processivity during intraflagellar transport. Stable localisation depends on the targeting of their docking proteins in the proximal zone. For ODA, this study characterized an ODA docking complex (ODA-DC) that is targeted directly to the proximal zone. Interestingly, the subunit composition of the ODA-DC in chordotonal neuron cilia appears to be different from the predicted ODA-DC in Drosophila sperm. 		Molina-Pelayo, C., Olguin, P., Mlodzik, M. and Glavic, A. (2022). The conserved Pelado/ZSWIM8 protein regulates actin dynamics by promoting linear actin filament polymerization. Life Sci Alliance 5(12). PubMed ID: 35940847
Summary:
Actin filament polymerization can be branched or linear, which depends on the associated regulatory proteins. Competition for actin monomers occurs between proteins that induce branched or linear actin polymerization. Cell specialization requires the regulation of actin filaments to allow the formation of cell type-specific structures, like cuticular hairs in Drosophila, formed by linear actin filaments. This study reports the functional analysis of CG34401/pelado, a gene encoding a SWIM domain-containing protein, conserved throughout the animal kingdom, called ZSWIM8 in mammals. Mutant pelado epithelial cells display actin hair elongation defects. This phenotype is reversed by increasing actin monomer levels or by either pushing linear actin polymerization or reducing branched actin polymerization. Similarly, in hemocytes, Pelado is essential to induce filopodia, a linear actin-based structure. This study further showed that this function of Pelado/ZSWIM8 is conserved in human cells, where Pelado inhibits branched actin polymerization in a cell migration context. In summary, these data indicate that the function of Pelado/ZSWIM8 in regulating actin cytoskeletal dynamics is conserved, favoring linear actin polymerization at the expense of branched filaments.
Lin, B., Luo, J. and Lehmann, R. (2022). An AMPK phosphoregulated RhoGEF feedback loop tunes cortical flow-driven amoeboid migration in vivo. Sci Adv 8(37): eabo0323. PubMed ID: 36103538
Summary:
Development, morphogenesis, immune system function, and cancer metastasis rely on the ability of cells to move through diverse tissues. To dissect migratory cell behavior in vivo, this study developed cell type-specific imaging and perturbation techniques for Drosophila primordial germ cells (PGCs). PGCs were found to use global, retrograde cortical actin flows for orientation and propulsion during guided developmental homing. PGCs use RhoGEF2, a RhoA-specific RGS-RhoGEF, as a dose-dependent regulator of cortical flow through a feedback loop requiring its conserved PDZ and PH domains for membrane anchoring and local RhoA activation. This feedback loop is regulated for directional migration by RhoGEF2 availability and requires AMPK rather than canonical Gα(12/13) signaling. AMPK multisite phosphorylation of RhoGEF2 near a conserved EB1 microtubule-binding SxIP motif releases RhoGEF2 from microtubule-dependent inhibition. Thus, this study established the mechanism by which global cortical flow and polarized RhoA activation can be dynamically adapted during natural cell navigation in a changing environment. 		Sturner, T., Ferreira Castro, A., Philipps, M., Cuntz, H. and Tavosanis, G. (2022). The branching code: A model of actin-driven dendrite arborization. Cell Rep 39(4): 110746. PubMed ID: 35476974
Summary:
The cytoskeleton is crucial for defining neuronal-type-specific dendrite morphologies. To explore how the complex interplay of actin-modulatory proteins (AMPs) can define neuronal types in vivo, this study focused on the class III dendritic arborization (c3da) neuron of Drosophila larvae. Using computational modeling, the main branches (MBs) of c3da neurons were demonstrated to follow general models based on optimal wiring principles, while the actin-enriched short terminal branches (STBs) require an additional growth program. To clarify the cellular mechanisms that define this second step, this study concentrated on STBs for an in-depth quantitative description of dendrite morphology and dynamics. Applying these methods systematically to mutants of six known and novel AMPs (Arp2/3, Capu, Ena, Singed, and Twinstar), the complementary roles were revealed of these individual AMPs in defining STB properties. These data suggest that diverse dendrite arbors result from a combination of optimal-wiring-related growth and individualized growth programs that are neuron-type specific.

Friday, November 18th - Enhancers and Transcriptional Regulation

Mazina, M. Y., Kovalenko, E. V., Evdokimova, A. A., Erokhin, M., Chetverina, D. and Vorobyeva, N. E. (2022). RNA Polymerase II "Pause" Prepares Promoters for Upcoming Transcription during Drosophila Development. Int J Mol Sci 23(18). PubMed ID: 36142573
Summary:
According to previous studies, during Drosophila embryogenesis, the recruitment of RNA polymerase II precedes active gene transcription. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. In addition, the composition of the RNA polymerase II "paused" complexes associated with promoters at different developmental stages are described in detail. For this purpose, ChIP-Seq analysis was performed using antibodies for various modifications of RNA polymerase II (total, Pol II CTD Ser5P, and Pol II CTD Ser2P) as well as for subunits of the NELF, DSIF, and PAF complexes and Brd4/Fs(1)h that control transcription elongation. It was found that during metamorphosis, similar to mid-embryogenesis, the promoters were bound by RNA polymerase II in the "paused" state, preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in a "pause" state was phosphorylated at Ser5 and Ser2 of Pol II CTD and bound the NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex included Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex was phosphorylated at Ser5 of Pol II CTD, but not at Ser2. These results indicate that, during mid-embryogenesis, RNA polymerase II stalls in the "post-pause" state, being phosphorylated at Ser2 of Pol II CTD (after the stage of p-TEFb action). During metamorphosis, the "pause" mechanism is closer to classical promoter-proximal pausing and is characterized by a low level of Pol II CTD Ser2P. 		Baltruk, L. J., Lavezzo, G. M., Machado-Lima, A., Digiampietri, L. A. and Andrioli, L. P. (2022). An additive repression mechanism sets the anterior limits of anterior pair-rule stripes 1. Cells Dev 171: 203802. PubMed ID: 35934285
Summary:
Segments are repeated anatomical units forming the body of insects. In Drosophila, the specification of the body takes place during the blastoderm through the segmentation cascade. Pair-rule genes such as hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) are of the intermediate level of the cascade and each pair-rule gene is expressed in seven transversal stripes along the antero-posterior axis of the embryo. Stripes are formed by independent cis-regulatory modules (CRMs) under the regulation of transcription factors of maternal source and of gap proteins of the first level of the cascade. The initial blastoderm of Drosophila is a syncytium and it also coincides with the mid-blastula transition when thousands of zygotic genes are transcribed and their products are able to diffuse in the cytoplasm. Thus, a complex regulation of the CRMs of the pair-rule stripes is anticipated. The CRMs of h 1, eve 1, run 1, ftz 1 are able to be activated by bicoid (bcd) throughout the anterior blastoderm and several lines of evidence indicate that they are repressed by the anterior gap genes slp1 (sloppy-paired 1), tll (tailless) and hkb (huckebein). The modest activity of these repressors led to the premise of a combinatorial mechanism regulating the expression of the CRMs of h 1, eve 1, run 1, ftz 1 in more anterior regions of the embryo. This possibility was tested by progressively removing the repression activities of slp1, tll and hkb. In doing so, it was possible to expose a mechanism of additive repression limiting the anterior borders of stripes 1. Stripes 1 respond depending on their distance from the anterior end and repressors operating at different levels.
Winant, M., Buhler, K., Clements, J., De Groef, S., Hens, K., Vulsteke, V. and Callaerts, P. (2022). Genome-wide analysis identifies Homothorax and Extradenticle as regulators of insulin in Drosophila Insulin-Producing cells. PLoS Genet 18(9): e1010380. PubMed ID: 36095003
Summary:
Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs publicly available transcriptome data of over 180 highly inbred fly lines were queried for dilp expression, and dilp expression was used as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. IPC-specific depletion of most genes was found to result in differences in expression of one or more of the dilps. Then on one of the candidate genes with the strongest effect on dilp expression, Homothorax, a transcription factor known for its role in eye development, was examined further. Homothorax and its binding partner Extradenticle were found to be involved in regulating dilp2, -3 and -5 expression; genetic depletion of both TFs shows phenotypes associated with reduced insulin signalling. Furthermore, evidence is provided that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, this study showed that this expression level-based GWAS approach identified genetic regulators implicated in IPC function and dilp expression. 		Wu, X., Bhatia, N., Grozinger, C. M. and Yi, S. V. (2022). Comparative studies of genomic and epigenetic factors influencing transcriptional variation in two insect species. G3 (Bethesda). PubMed ID: 36137211
Summary:
Different genes show different levels of expression variability. For example, highly expressed genes tend to exhibit less expression variability. Genes whose promoters have TATA box and initiator motifs tend to have increased expression variability. On the other hand, DNA methylation of transcriptional units, or gene body DNA methylation, is associated with reduced gene expression variability in many species. Interestingly, some insect lineages, most notably Diptera including the canonical model insect Drosophila melanogaster, have lost DNA methylation. Therefore, it is of interest to determine whether genomic features similarly influence gene expression variability in lineages with and without DNA methylation. This study analyzed recently generated large-scale data sets in D. melanogaster and honey bee (Apis mellifera) to investigate these questions. This analysis shows that increased gene expression levels are consistently associated with reduced expression variability in both species, while the presence of TATA box is consistently associated with increased gene expression variability. In contrast, initiator motifs and gene lengths have weak effects limited to some data sets. Importantly, this study showed that sequence characteristics indicative of gene body DNA methylation is strongly and negatively associate with gene expression variability in honey bees, while it shows no such association in D. melanogaster. These results suggest the evolutionary loss of DNA methylation in some insect lineages has reshaped the molecular mechanisms concerning the regulation of gene expression variability.
Zielke, N., Vaharautio, A., Liu, J., Kivioja, T. and Taipale, J. (2022). Upregulation of ribosome biogenesis via canonical E-boxes is required for Myc-driven proliferation. Dev Cell 57(8): 1024-1036. PubMed ID: 35472319
Summary:
The transcription factor Myc drives cell growth across animal phyla and is activated in most forms of human cancer. However, it is unclear which Myc target genes need to be regulated to induce growth and whether multiple targets act additively or if induction of each target is individually necessary. This study identified Myc target genes whose regulation is conserved between humans and flies; Myc-binding sites (E-boxes) were deleted in the promoters of fourteen of these genes in Drosophila. E-box mutants of essential genes were homozygous viable, indicating that the E-boxes are not required for basal expression. Eight E-box mutations led to Myc-like phenotypes; the strongest mutant, ppanEbox-/-, also made the flies resistant to Myc-induced cell growth without affecting Myc-induced apoptosis. The ppanEbox-/- flies are healthy and display only a minor developmental delay, suggesting that it may be possible to treat or prevent tumorigenesis by targeting individual downstream targets of Myc. 		Wylie, A., Jones, A. E., Das, S., Lu, W. J. and Abrams, J. M. (2022). Distinct p53 isoforms code for opposing transcriptional outcomes. Dev Cell 57(15): 1833-1846. PubMed ID: 35820415
Summary:
p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. This study investigated synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control in vivo. Transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

Thursday November 17th - Enzyme Characterization

Zhu, W., Duan, Y., Chen, J., Merzendorfer, H., Zou, X. and Yang, Q. (2022). SERCA interacts with chitin synthase and participates in cuticular chitin biogenesis in Drosophila. Insect Biochem Mol Biol 145: 103783. PubMed ID: 35525402
Summary:
The biogenesis of chitin, a major structural polysaccharide found in the cuticle and peritrophic matrix, is crucial for insect growth and development. Chitin synthase, a membrane-integral β-glycosyltransferase, has been identified as the core of the chitin biogenesis machinery. However, a yet unknown number of auxiliary proteins appear to assist in chitin biosynthesis, whose precise function remains elusive. This study identified a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), in the fruit fly Drosophila melanogaster, as a chitin biogenesis-associated protein. The physical interaction between DmSERCA and epidermal chitin synthase (Krotzkopf verkehrt, Kkv) was demonstrated and analyzed using split-ubiquitin membrane yeast two-hybrid, bimolecular fluorescent complementation, pull-down, and immunoprecipitation assays. The interaction involves N-terminal regions (aa 48-81 and aa 247-33) and C-terminal regions (aa 743-783 and aa 824-859) of DmSERCA and two N-terminal regions (aa 121-179 and aa 369-539) of Kkv, all of which are predicted be transmembrane helices. While tissue-specific knock-down of DmSERCA in the epidermis caused larval and pupal lethality, the knock-down of DmSERCA in wings resulted in smaller and crinkled wings, a significant decrease in chitin deposition, and the loss of chitin lamellar structure. Although DmSERCA is well-known for its role in muscular contraction, this study reveals a novel role in chitin synthesis, contributing to knowledge on the machinery of chitin biogenesis. 		Porcellato, E., Gonzalez-Sanchez, J. C., ..., Wieland, F. T. and Metzendorf, C. (2022). The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes. PLoS One 17(8): e0261543. PubMed ID: 35960718
Summary:
S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. This study presents the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs), DHHC-BioID, a proximity biotinylation-based method, was developed. In S2R+ cells, ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, this study showed that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary this study provides the first systematic analysis of a Drosophila palmitoylome. DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, this study established DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.
Kawakami, J., Brooks, D., Zalmai, R., Hartson, S. D., Bouyain, S. and Geisbrecht, E. R. (2022). Complex protein interactions mediate Drosophila Lar function in muscle tissue. PLoS One 17(5): e0269037. PubMed ID: 35622884
Summary:
The type IIa family of receptor protein tyrosine phosphatases (RPTPs), including Lar, RPTPσ and RPTPδ, are well-studied in coordinating actin cytoskeletal rearrangements during axon guidance and synaptogenesis. To determine whether this regulation is conserved in other tissues, interdisciplinary approaches were utilized to study Lar-RPTPs in the Drosophila musculature. This study found that the single fly ortholog, Drosophila Lar (Dlar), is localized to the muscle costamere and that a decrease in Dlar causes aberrant sarcomeric patterning, deficits in larval locomotion, and integrin mislocalization. Sequence analysis uncovered an evolutionarily conserved Lys-Gly-Asp (KGD) signature in the extracellular region of Dlar. Since this tripeptide sequence is similar to the integrin-binding Arg-Gly-Asp (RGD) motif, the hypothesis was tested that Dlar directly interacts with integrin proteins. However, structural analyses of the fibronectin type III domains of Dlar and two vertebrate orthologs that include this conserved motif indicate that this KGD tripeptide is not accessible and thus unlikely to mediate physical interactions with integrins. These results, together with the proteomics identification of basement membrane (BM) proteins as potential ligands for type IIa RPTPs, suggest a complex network of protein interactions in the extracellular space that may mediate Lar function and/or signaling in muscle tissue. 		Fabre, B., Choteau, S. A., Duboe, C., Pichereaux, C., Montigny, A., Korona, D., Deery, M. J., Camus, M., Brun, C., Burlet-Schiltz, O., Russell, S., Combier, J. P., Lilley, K. S. and Plaza, S. (2022). In Depth Exploration of the Alternative Proteome of Drosophila melanogaster. Front Cell Dev Biol 10: 901351. PubMed ID: 35721519
Summary:
Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. The article describes a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, 14 alternative proteins were observed with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins.
Huang, Y., Li, L. and Rong, Y. S. (2022). JiangShi (僵尸): a widely distributed Mucin-like protein essential for Drosophila development. G3 (Bethesda). PubMed ID: 35595239
Summary:
Epithelia exposed to elements of the environment are protected by a mucus barrier in mammals. This barrier also serves to lubricate during organ movements and to mediate substance exchanges between the environmental milieu and internal organs. A major component of the mucus barrier is a class of glycosylated proteins called Mucin. Mucin and mucin-related proteins are widely present in the animal kingdom. Mucin mis-regulation has been reported in many diseases such as cancers and ones involving the digestive and respiratory tracts. Although the biophysical properties of isolated Mucins have been extensively studied, in vivo models remain scarce for the study of their functions and regulations. This study characterize the Mucin-like JiangShi (JS) protein and its mutations in the fruit fly Drosophila. JS is an extracellular glycoprotein with domain features reminiscent of mammalian non-membranous Mucins, and one of the most widely distributed Mucin-like proteins studied in Drosophila. Both loss and over-production of JS lead to terminal defects in adult structures and organismal death. Although the physiological function of JS remains poorly defined, a genetically tractable model system is presented for the in vivo studies of Mucin-like molecules. 		Liu, Y., Chen, Z., Wang, Z. H., Delaney, K. M., Tang, J., Pirooznia, M., Lee, D. Y., Tunc, I., Li, Y. and Xu, H. (2022). The PPR domain of mitochondrial RNA polymerase is an exoribonuclease required for mtDNA replication in Drosophila melanogaster. Nat Cell Biol 24(5): 757-765. PubMed ID: 35449456
Summary:
Mitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase is thought to be the primase for mtDNA replication. However, it is unclear how this enzyme, which normally transcribes long polycistronic RNAs, can produce short RNA oligonucleotides to initiate mtDNA replication. This study shows that the PPR domain of Drosophila mitochondrial RNA polymerase (PolrMT) has 3'-to-5' exoribonuclease activity, which is indispensable for PolrMT to synthesize short RNA oligonucleotides and prime DNA replication in vitro. An exoribonuclease-deficient mutant, PolrMTE423P, partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in PolrMT-mutant flies, indicating that the exoribonuclease activity is necessary for mtDNA replication. In addition, overexpression of PolrMT(E423P) in adult flies leads to severe neuromuscular defects and a marked increase in mtDNA transcript errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription.

Wednesday November 16th - Adult Neural Development and Function

Alpert, M. H., Gil, H., Para, A. and Gallio, M. (2022). A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat. Curr Biol. PubMed ID: 35981537
Summary:
Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. This study use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons(1)) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ∼25°C (i.e., "hot" temperature). We identify ACs in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.(2) LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Previous work described a distinct thermometer circuit for cold temperature.(3) Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales. 		Baker, C. A., McKellar, C., Pang, R., Nern, A., Dorkenwald, S., Pacheco, D. A., Eckstein, N., Funke, J., Dickson, B. J. and Murthy, M. (2022). Neural network organization for courtship-song feature detection in Drosophila. Curr Biol 32(15): 3317-3333.e3317. PubMed ID: 35793679
Summary:
Animals communicate using sounds in a wide range of contexts, and auditory systems must encode behaviorally relevant acoustic features to drive appropriate reactions. How feature detection emerges along auditory pathways has been difficult to solve due to challenges in mapping the underlying circuits and characterizing responses to behaviorally relevant features. This paper describes auditory activity in the Drosophila melanogaster brain and investigated feature selectivity for the two main modes of fly courtship song, sinusoids and pulse trains. Twenty-four new cell types of the intermediate layers of the auditory pathway were identified, and using a new connectomic resource, FlyWire, all synaptic connections between these cell types, in addition to connections to known early and higher-order auditory neurons were mapped-this represents the first circuit-level map of the auditory pathway. The sign (excitatory or inhibitory) was determined of most synapses in this auditory connectome. Auditory neurons were found to display a continuum of preferences for courtship song modes and that neurons with different song-mode preferences and response timescales are highly interconnected in a network that lacks hierarchical structure. Nonetheless, this study found that the response properties of individual cell types within the connectome are predictable from their inputs. This study thus provides new insights into the organization of auditory coding within the Drosophila brain.
Krishnamurthy, K., Hermundstad, A. M., Mora, T., Walczak, A. M. and Balasubramanian, V. (2022). Disorder and the Neural Representation of Complex Odors. Front Comput Neurosci 16: 917786. PubMed ID: 36003684
Summary:
Animals smelling in the real world use a small number of receptors to sense a vast number of natural molecular mixtures, and proceed to learn arbitrary associations between odors and valences. This study proposes how the architecture of olfactory circuits leverages disorder, diffuse sensing and redundancy in representation to meet these immense complementary challenges. First, the diffuse and disordered binding of receptors to many molecules compresses a vast but sparsely-structured odor space into a small receptor space, yielding an odor code that preserves similarity in a precise sense. Introducing any order/structure in the sensing degrades similarity preservation. Next, lateral interactions further reduce the correlation present in the low-dimensional receptor code. Finally, expansive disordered projections from the periphery to the central brain reconfigure the densely packed information into a high-dimensional representation, which contains multiple redundant subsets from which downstream neurons can learn flexible associations and valences. Moreover, introducing any order in the expansive projections degrades the ability to recall the learned associations in the presence of noise. This theory was tested empirically using data from Drosophila. The theory suggests that the neural processing of sparse but high-dimensional olfactory information differs from the other senses in its fundamental use of disorder. 		Hayashi, T. T., MacKenzie, A. J., Ganguly, I., Ellis, K. E., Smihula, H. M., Jacob, M. S., Litwin-Kumar, A. and Caron, S. J. C. (2022). Mushroom body input connections form independently of sensory activity in Drosophila melanogaster. Curr Biol. PubMed ID: 35977547
Summary:
Associative brain centers, such as the insect mushroom body, need to represent sensory information in an efficient manner. In Drosophila melanogaster, the Kenyon cells of the mushroom body integrate inputs from a random set of olfactory projection neurons, but some projection neurons-namely those activated by a few ethologically meaningful odors-connect to Kenyon cells more frequently than others. This biased and random connectivity pattern is conceivably advantageous, as it enables the mushroom body to represent a large number of odors as unique activity patterns while prioritizing the representation of a few specific odors. How this connectivity pattern is established remains largely unknown. This study tested whether the mechanisms patterning the connections between Kenyon cells and projection neurons depend on sensory activity or whether they are hardwired. A large number of mushroom body input connections were mapped in partially anosmic flies-flies lacking the obligate odorant co-receptor Orco-and in wild-type flies. Statistical analyses of these datasets reveal that the random and biased connectivity pattern observed between Kenyon cells and projection neurons forms normally in the absence of most olfactory sensory activity. This finding supports the idea that even comparatively subtle, population-level patterns of neuronal connectivity can be encoded by fixed genetic programs and are likely to be the result of evolved prioritization of ecologically and ethologically salient stimuli.
Matheson, A. M. M., Lanz, A. J., Medina, A. M., Licata, A. M., Currier, T. A., Syed, M. H. and Nagel, K. I. (2022). A neural circuit for wind-guided olfactory navigation. Nat Commun 13(1): 4613. PubMed ID: 35941114
Summary:
To navigate towards a food source, animals frequently combine odor cues about source identity with wind direction cues about source location. Where and how these two cues are integrated to support navigation is unclear. This study describes a pathway to the Drosophila fan-shaped body that encodes attractive odor and promotes upwind navigation. We show that neurons throughout this pathway encode odor, but not wind direction. Using connectomics, fan-shaped body local neurons called hΔC that receive input from this odor pathway and a previously described wind pathway. hΔC neurons exhibit odor-gated, wind direction-tuned activity, that sparse activation of h∆C neurons promotes navigation in a reproducible direction, and that hΔC activity is required for persistent upwind orientation during odor. Based on connectome data, a computational model was developed showing how hΔC activity can promote navigation towards a goal such as an upwind odor source. The results suggest that odor and wind cues are processed by separate pathways and integrated within the fan-shaped body to support goal-directed navigation. 		Park, A., Croset, V., Otto, N., Agarwal, D., Treiber, C. D., Meschi, E., Sims, D. and Waddell, S. (2022). Gliotransmission of D-serine promotes thirst-directed behaviors in Drosophila. Curr Biol. PubMed ID: 35963239
Summary:
Thirst emerges from a range of cellular changes that ultimately motivate an animal to consume water. Although thirst-responsive neuronal signals have been reported, the full complement of brain responses is unclear. This study identified molecular and cellular adaptations in the brain using single-cell sequencing of water-deprived Drosophila. Water deficiency primarily altered the glial transcriptome. Screening the regulated genes revealed astrocytic expression of the astray-encoded phosphoserine phosphatase to bi-directionally regulate water consumption. Astray synthesizes the gliotransmitter D-serine, and vesicular release from astrocytes is required for drinking. Moreover, dietary D-serine rescues astray-dependent drinking deficits while facilitating water consumption and expression of water-seeking memory. D-serine action requires binding to neuronal NMDA-type glutamate receptors. Fly astrocytes contribute processes to tripartite synapses, and the proportion of astrocytes that are themselves activated by glutamate increases with water deprivation. It is proposed that thirst elevates astrocytic D-serine release, which awakens quiescent glutamatergic circuits to enhance water procurement.

Tuesday, November 15th - Evolution

Marcella, M., Piacentini, L., Berloco, M. F., Casale, A. M., Cappucci, U., Pimpinelli, S. and Fanti, L. (2022). Cytological heterogeneity of heterochromatin among 10 sequenced Drosophila species. Genetics. PubMed ID: 35946576
Summary:
In Drosophila chromosomal rearrangements can be maintained and are associated with karyotypic variability among populations from different geographic localities. The abundance of variability in gene arrangements among chromosomal arms is even greater when comparing more distantly related species and the study of these chromosomal changes has provided insights into the evolutionary history of species in the genus. Additionally, the sequencing of genomes of several Drosophila species has offered the opportunity to establish the global pattern of genomic evolution, at both genetic and chromosomal level. The combined approaches of comparative analysis of syntenic blocks and direct physical maps on polytene chromosomes have elucidated changes in the orientation of genomic sequences and the difference between heterochromatic and euchromatic regions. Unfortunately, the centromeric heterochromatic regions cannot be studied using the cytological maps of polytene chromosomes because they are underreplicated and therefore reside in the chromocenter. In D. melanogaster, a cytological map of the heterochromatin has been elaborated using mitotic chromosomes from larval neuroblasts. The current work has expanded on that mapping by producing cytological maps of the mitotic heterochromatin in an additional 10 sequenced Drosophila species. These maps highlight two apparently different paths, for the evolution of the pericentric heterochromatin between the subgenera Sophophora and Drosophila. One path leads towards a progressive complexity of the pericentric heterochromatin (Sophofora) and the other towards a progressive simplification (Drosophila). These maps are also useful for a better understanding how karyotypes have been altered by chromosome arm reshuffling during evolution. 		Aggarwal, D. D., Mishra, P. and Singh, M. (2022). An analysis of direct and indirect effects in Drosophila melanogaster undergoing a few cycles of experimental evolution for stress-related traits. Comp Biochem Physiol B Biochem Mol Biol 263: 110795. PubMed ID: 35970341
Summary:
The physiological mechanisms underpinning adaptations to starvation and cold stresses have been extensively studied in Drosophila, yet the understanding of correlated changes in stress-related and life-history traits, as well as the energetics of stress tolerance, still remains elusive. To answer the questions empirically in this context, this study allowed D. melanogaster to evolve for either increased starvation or cold tolerance (24-generations / regime) in an experimental evolution system, and examined whether selection of either trait affects un-selected stress trait, as well as the impacts potential changes in life-history and mating success-related traits. The results revealed remarkable changes in starvation/cold tolerance (up to 1.5-fold) as a direct effect of selection, while cold tolerance had been dramatically reduced (1.26-fold) in the starvation tolerant (ST) lines compared to control counterparts, although no such changes were evident in cold-tolerant (CT) lines. ST lines exhibited a higher level of body lipids and a reduced level of trehalose content, while CT lines accumulated a greater levels of body lipid and trehalose contents. Noticeably, it was found that selection for starvation or cold tolerance positively correlates with larval development time, longevity, and copulation duration, indicating that these traits are among the most common targets of selection trajectories shaping stress tolerance. Altogether, this study highlights the complexity of mechanisms evolved in ST lines that contribute to enhanced starvation tolerance, but also negatively impact cold tolerance. Nevertheless, mechanisms forging enhanced cold tolerance in CT lines appear not to target starvation tolerance. Moreover, the parallel changes in life history/mating success traits across stress regimes could indicate some generic pathways evolved in stressful environments, targeting life-history and mating success characteristics to optimize fitness.
Feitzinger, A. A., Le, A., Thompson, A., Haseeb, M., Murugesan, M. K., Tang, A. M. and Lott, S. E. (2022). Natural variation in the maternal and zygotic mRNA complements of the early embryo in Drosophila melanogaster. BMC Genomics 23(1): 641. PubMed ID: 36076188
Summary:
Maternal gene products supplied to the egg during oogenesis drive the earliest events of development in all metazoans. After the initial stages of embryogenesis, maternal transcripts are degraded as zygotic transcription is activated; this is known as the maternal to zygotic transition (MZT). Recently, it has been shown that the expression of maternal and zygotic transcripts have evolved in the Drosophila genus over the course of 50 million years. However, the extent of natural variation of maternal and zygotic transcripts within a species has yet to be determined. This study asked how the maternal and zygotic pools of mRNA vary within and between populations of D. melanogaster. In order to maximize sampling of genetic diversity, African lines of D. melanogaster originating from Zambia as well as DGRP lines originating from North America were chosen for transcriptomic analysis. Generally, it was found that maternal transcripts are more highly conserved, and zygotic transcripts evolve at a higher rate. There is more within-population variation in transcript abundance than between populations, and expression variation is highest post- MZT between African lines. Determining the natural variation of gene expression surrounding the MZT in natural populations of D. melanogaster gives insight into the extent of how a tightly regulated process may vary within a species, the extent of developmental constraint at both stages and on both the maternal and zygotic genomes, and reveals expression changes allowing this species to adapt as it spread across the world. 		Hine, E., Runcie, D. E., Allen, S. L., Wang, Y., Chenoweth, S. F., Blows, M. W. and McGuigan, K. (2022). Maintenance of quantitative genetic variance in complex, multi-trait phenotypes: The contribution of rare, large effect variants in two Drosophila species. Genetics. PubMed ID: 35961029
Summary:
The interaction of evolutionary processes to determine quantitative genetic variation has implications for contemporary and future phenotypic evolution, as well as for our ability to detect causal genetic variants. While theoretical studies have provided robust predictions to discriminate among competing models, empirical assessment of these has been limited. In particular, theory highlights the importance of pleiotropy in resolving observations of selection and mutation, but empirical investigations have typically been limited to few traits. This study applied high dimensional Bayesian Sparse Factor Genetic modelling to gene expression datasets in two species, Drosophila melanogaster and D. serrata, to explore the distributions of genetic variance across high-dimensional phenotypic space. Surprisingly, most of the heritable trait covariation was due to few lines (genotypes) with extreme (>3 interquartile ranges from the median) values. Intriguingly, while genotypes extreme for a multivariate factor also tended to have a higher proportion of individual traits that were extreme, genotypes were also observed that were extreme for multivariate factors but not for any individual trait. Other consistent differences between heritable multivariate factors with outlier lines vs. those factors without extreme values, including differences in gene functions. These observations were used to identify further data required to advance our understanding of the evolutionary dynamics and nature of standing genetic variation for quantitative traits.
Moutinho, A. F., Eyre-Walker, A. and Dutheil, J. Y. (2022). Strong evidence for the adaptive walk model of gene evolution in Drosophila and Arabidopsis. PLoS Biol 20(9): e3001775. PubMed ID: 36099311
Summary:
Understanding the dynamics of species adaptation to their environments has long been a central focus of the study of evolution. Theories of adaptation propose that populations evolve by "walking" in a fitness landscape. This "adaptive walk" is characterised by a pattern of diminishing returns, where populations further away from their fitness optimum take larger steps than those closer to their optimal conditions. Hence, it is expected that young genes evolve faster and experience mutations with stronger fitness effects than older genes because they are further away from their fitness optimum. Testing this hypothesis, however, constitutes an arduous task. Young genes are small, encode proteins with a higher degree of intrinsic disorder, are expressed at lower levels, and are involved in species-specific adaptations. Since all these factors lead to increased protein evolutionary rates, they could be masking the effect of gene age. While controlling for these factors, this study used population genomic data sets of Arabidopsis and Drosophila and estimated the rate of adaptive substitutions across genes from different phylostrata. A gene's evolutionary age was found to significantly impact the molecular rate of adaptation. Moreover, it was observed that substitutions in young genes tend to have larger physicochemical effects. This study, therefore, provides strong evidence that molecular evolution follows an adaptive walk model across a large evolutionary timescale. 		Yu, Y. and Bergland, A. O. (2022). Distinct signals of clinal and seasonal allele frequency change at eQTLs in Drosophila melanogaster. Evolution. PubMed ID: 36097359
Summary:
Populations of short-lived organisms can respond to spatial and temporal environmental heterogeneity through local adaptation. Local adaptation can be reflected on both phenotypic and genetic levels, and it has been documented in many organisms. Although complex fitness-related phenotypes have been shown to vary across latitudinal clines and seasons in similar ways in Drosophila melanogaster populations, the comparative signals of local adaptation across space and time remain poorly understood. This study examined patterns of allele frequency change across a latitudinal cline and between seasons at previously reported expression quantitative trait loci (eQTLs). eQTLs were divided into groups by using differential expression profiles of fly populations collected across latitudinal clines or exposed to different environmental conditions. In general, eQTLs were found to be enriched for clinally varying polymorphisms, and these eQTLs changed in frequency in concordant ways across the cline and in response to starvation and chill-coma. The enrichment of eQTLs among seasonally varying polymorphisms is more subtle, and the direction of allele frequency change at eQTLs appears to be somewhat idiosyncratic. Taken together, it is suggested that clinal adaptation at eQTLs is at least partially distinct from seasonal adaptation.

Monday November 14th - Disease models

Chen, Z., Cordero, J., Alqarni, A. M., Slack, C., Zeidler, M. P. and Bellantuono, I. (2022). Zoledronate Extends Health Span and Survival via the Mevalonate Pathway in a FOXO-dependent Manner. J Gerontol A Biol Sci Med Sci 77(8): 1494-1502. PubMed ID: 34137822
Summary:
Over recent decades, increased longevity has not been paralleled by extended health span, resulting in more years spent with multiple diseases in older age. As such, interventions to improve health span are urgently required. Zoledronate (Zol) is a nitrogen-containing bisphosphonate, which inhibits the farnesyl pyrophosphate synthase enzyme, central to the mevalonate pathway. It is already used clinically to prevent fractures in osteoporotic patients, who have been reported to derive unexpected and unexplained survival benefits. Using Drosophila as a model the effects were determined of Zol on life span, parameters of health span (climbing ability and intestinal dysplasia), and the ability to confer resistance to oxidative stress using a combination of genetically manipulated Drosophila strains and Western blotting. This study study shows that Zol extended life span, improved climbing activity, and reduced intestinal epithelial dysplasia and permeability with age. Mechanistic studies showed that Zol conferred resistance to oxidative stress and reduced accumulation of X-ray-induced DNA damage via inhibition of farnesyl pyrophosphate synthase. Moreover, Zol was associated with inhibition of phosphorylated AKT in the mammalian target of rapamycin pathway downstream of the mevalonate pathway and required dFOXO for its action, both molecules associated with increased longevity. Taken together, this work indicates that Zol, a drug already widely used to prevent osteoporosis and dosed only once a year, modulates important mechanisms of aging. Its repurposing holds great promise as a treatment to improve health span. 		Fu, B., Ma, R., Liu, F., Chen, X., Teng, X., Yang, P., Liu, J., Zhao, D. and Sun, L. (2022). Ginsenosides improve reproductive capability of aged female Drosophila through mechanism dependent on ecdysteroid receptor (ECR) and steroid signaling pathway. Front Endocrinol (Lausanne) 13: 964069. PubMed ID: 36017314
Summary:
Aging ovaries caused diminished fertility and depleted steroid hormone level. Ginsenosides, the active ingredient in ginseng, had estrogen-like hormonal effects. This study found that ginsenosides improved the quantity and quality of the offspring, prolonged life and restored muscle ability in aged female Drosophila. In addition, ginsenosides inhibited ovarian atrophy and maintained steroid hormone 20-Hydroxyecdysone (20E) and juvenile-preserving hormone (JH)) levels. Ginsenosides activated ecdysteroid receptor (ECR) and increased the expression of the early transcription genes E74 and Broad (Br), which triggered steroid signaling pathway. Meanwhile, ginsenosides promoted JH biosynthesis by increasing the expression of Hydroxyl-methylglutaryl-CoA reductase (HMGR) and juvenile hormone acid O-methyltransferase (JHAMT). Subsequently, JH was bound to Methoprene Tolerant (Met) and activated the transcription of the responsive gene Kruppel Homolog 1 (Kr-h1), which coordinated with 20E signaling to promote the reproduction of aged female Drosophila. The reproductive capacity and steroid hormone levels were not improved and the steroid signaling pathway was not activated in ginsenoside-treated ECR knockout Drosophila. This suggested that ginsenosides played a role dependent on targeted ECR. Furthermore, 17 kinds of ginsenoside monomers were identified from the total ginsenosides. Among them, Rg1, Re and Rb1 improved the reproductive capacity and steroid hormone levels of aged female Drosophila, which has similar effects to the total ginsenoside. These results indicated that ginsenosides could enhance the reproductive capacity of aged female Drosophila by activating steroid signals dependent on nuclear receptor ECR. In addition, ginsenoside monomers Rg1, Rb1 and Re are the main active components of total ginsenosides to improve reproductive ability. This will provide strong evidence that ginsenosides had the potential to alleviate age-induced reproductive degradation.
Yin, Y., Ma, P., Wang, S., Zhang, Y., Han, R., Huo, C., Wu, M. and Deng, H. (2022). The CRTC-CREB axis functions as a transcriptional sensor to protect against proteotoxic stress in Drosophila. Cell Death Dis 13(8): 688. PubMed ID: 35933423
Summary:
cAMP Responsible Element Binding Protein (CREB) is an evolutionarily conserved transcriptional factor that regulates cell growth, synaptic plasticity and so on. This study unexpectedly found proteasome inhibitors, such as MLN2238, robustly increase CREB activity in adult flies through a large-scale compound screening. Mechanistically, reactive oxidative species (ROS) generated by proteasome inhibition are required and sufficient to promote CREB activity through c-Jun N-terminal kinase (JNK). In 293 T cells, JNK activation by MLN2238 is also required for increase of CREB phosphorylation at Ser(133). Meanwhile, transcriptome analysis in fly intestine identified a group of genes involved in redox and proteostatic regulation are augmented by overexpressing CRTC (CREB-regulated transcriptional coactivator). Intriguingly, CRTC overexpression in muscles robustly restores protein folding and proteasomal activity in a fly Huntington's disease (HD) model, and ameliorates HD related pathogenesis, such as protein aggregates, motility, and lifespan. Moreover, CREB activity increases during aging, and further enhances its activity can suppress protein aggregates in aged muscles. Together, these results identified CRTC/CREB downstream ROS/JNK signaling as a conserved sensor to tackle oxidative and proteotoxic stresses. Boosting CRTC/CREB activity is a potential therapeutic strategy to treat aging related protein aggregation diseases. 		Deng, N., Wu, Y. Y., Feng, Y., Hsieh, W. C., Song, J. S., Lin, Y. S., Tseng, Y. H., Liao, W. J., Chu, Y. F., Liu, Y. C., Chang, E. C., Liu, C. R., Sheu, S. Y., Su, M. T., Kuo, H. C., Cohen, S. N. and Cheng, T. H. (2022). Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes. Proc Natl Acad Sci U S A 119(32): e2204779119. PubMed ID: 35914128
Summary:
Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, this study identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction, and then their effects were investigated on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). This study reports that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. These findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.
Malacrino, A., Brengdahl, M. I., Kimber, C. M., Mital, A., Shenoi, V. N., Mirabello, C. and Friberg, U. (2022). Ageing desexualizes the Drosophila brain transcriptome. Proc Biol Sci 289(1980): 20221115. PubMed ID: 35946149
Summary:
General evolutionary theory predicts that individuals in low condition should invest less in sexual traits compared to individuals in high condition. Whether this positive association between condition and investment also holds between young (high condition) and senesced (low condition) individuals is however less clear, since elevated investment into reproduction may be beneficial when individuals approach the end of their life. To address how investment into sexual traits changes with age, genes were studied with sex-biased expression in the brain, the tissue from which sexual behaviours are directed. Across two distinct populations of Drosophila melanogaster, it was found that old brains display fewer sex-biased genes, and that expression of both male-biased and female-biased genes converges towards a sexually intermediate phenotype owing to changes in both sexes with age. It was further found that sex-biased genes in general show heightened age-dependent expression in comparison to unbiased genes and that age-related changes in the sexual brain transcriptome are commonly larger in males than females.The results hence show that ageing causes a desexualization of the fruit fly brain transcriptome and that this change mirrors the general prediction that low condition individuals should invest less in sexual phenotypes. 		Gagliani, E. K., Gutzwiller, L. M., Kuang, Y., Odaka, Y., Hoffmeister, P., Hauff, S., Turkiewicz, A., Harding-Theobald, E., Dolph, P. J., Borggrefe, T., Oswald, F., Gebelein, B. and Kovall, R. A. (2022). A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects. PLoS Genet 18(8): e1010335. PubMed ID: 35951645
Summary:
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. This study found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

Thursday, November10th - Cell cycle

Hecht, S., Perez-Mockus, G., Schienstock, D., Recasens-Alvarez, C., Merino-Aceituno, S., Smith, M., Salbreux, G., Degond, P. and Vincent, J. P. (2022). Mechanical constraints to cell-cycle progression in a pseudostratified epithelium. Curr Biol 32(9): 2076-2083. PubMed ID: 35338851
Summary:
As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination, mechanical feedback, perhaps via adherens junctions, has been suggested to play a role. However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture. In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM), which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis. To explore how mechanical constraints affect IKNM, an individual-based model was devised that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which were validated with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). However, this model does not preclude indefinite growth, leading to a postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, the updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth. 		Mar, J., Makhijani, K., Flaherty, D. and Bhat, K. M. (2022). Nuclear Prospero allows one-division potential to neural precursors and post-mitotic status to neurons via opposite regulation of Cyclin E. PLoS Genet 18(8): e1010339. PubMed ID: 35939521
Summary:
In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. This study shows that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization.
Ranjan, R., Snedeker, J., Wooten, M., Chu, C., Bracero, S., Mouton, T. and Chen, X. (2022). Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage. Dev Cell 57(9): 1102-1118.e1107. PubMed ID: 35483360
Summary:
During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. this study tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. Unique features were found that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination. 		Wong, S. S., Wilmott, Z. M., Saurya, S., Alvarez-Rodrigo, I., Zhou, F. Y., Chau, K. Y., Goriely, A. and Raff, J. W. (2022). Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly. Embo j 41(11): e110891. PubMed ID: 35505659
Summary:
Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. This study shows that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process-peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. It is proposed that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.
Darnat, P., Burg, A., Salle, J., Lacoste, J., Louvet-Vallee, S., Gho, M. and Audibert, A. (2022). Cortical Cyclin A controls spindle orientation during asymmetric cell divisions in Drosophila. Nat Commun 13(1): 2723. PubMed ID: 35581185
Summary:
The coordination between cell proliferation and cell polarity is crucial to orient the asymmetric cell divisions to generate cell diversity in epithelia. In many instances, the Frizzled/Dishevelled planar cell polarity pathway is involved in mitotic spindle orientation, but how this is spatially and temporally coordinated with cell cycle progression has remained elusive. Using Drosophila sensory organ precursor cells as a model system, this study shows that Cyclin A, the main Cyclin driving the transition to M-phase of the cell cycle, is recruited to the apical-posterior cortex in prophase by the Frizzled/Dishevelled complex. This cortically localized Cyclin A then regulates the orientation of the division by recruiting Mud, a homologue of NuMA, the well-known spindle-associated protein. The observed non-canonical subcellular localization of Cyclin A reveals this mitotic factor as a direct link between cell proliferation, cell polarity and spindle orientation. 		Steinacker, T. L., Wong, S. S., Novak, Z. A., Saurya, S., Gartenmann, L., van Houtum, E. J. H., Sayers, J. R., Lagerholm, B. C. and Raff, J. W. (2022). Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL. J Cell Biol 221(9). PubMed ID: 35861803
Summary:
Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. This study shows that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis.

Wednesday November 9th - Larval and Adult Development

Dong, W., Flaven-Pouchon, J., Gao, Y. H., Song, C. Y., Wakil, A. E., Zhang, J. Z. and Moussian, B. (2022). Chitinase 6 is required for procuticle thickening and organ shape in Drosophila wing. Insect Sci. PubMed ID: 36114809
Summary:
The polysaccharide chitin is a major scaffolding molecule in the insect cuticle. In order to be functional, both chitin amounts and chitin organization have been shown to be important parameters. Despite great advances in the past decade, the molecular mechanisms of chitin synthesis and organization are not fully understood. This study has characterized the function of the Chitinase 6 (Cht6) in the formation of the wing, which is a simple flat cuticle organ, in the fruit fly Drosophila melanogaster. Reduction of Cht6 function by RNA interference during wing development does not affect chitin organization, but entails a thinner cuticle suggesting reduced chitin amounts. This phenotype is opposed to the one reported recently to be caused by reduction of Cht10 expression. Probably as a consequence, cuticle permeability to xenobiotics is enhanced in Cht6-less wings. Massive deformation of these wings was also observed. In addition, the shape of the abdomen is markedly changed upon abdominal suppression of Cht6. Finally, it was found that suppression of Cht6 transcript levels influences the expression of genes coding for enzymes of the chitin biosynthesis pathway. This finding indicates that wing epidermal cells respond to activity changes of Cht6 probably trying to adjust chitin amounts. Together, in a working model, it is proposed that Cht6-introduced modifications of chitin are needed for chitin synthesis to proceed correctly. Cuticle thickness, according to this hypothesis, is in turn required for correct organ or body part shape. The molecular mechanisms of this processes shall be characterized in the future. 		Worley, M. I., Everetts, N. J., Yasutomi, R., Chang, R. J., Saretha, S., Yosef, N. and Hariharan, I. K. (2022). Ets21C sustains a pro-regenerative transcriptional program in blastema cells of Drosophila imaginal discs. Curr Biol 32(15): 3350-3364. PubMed ID: 35820420
Summary:
An important unanswered question in regenerative biology is to what extent regeneration is accomplished by the reactivation of gene regulatory networks used during development versus the activation of regeneration-specific transcriptional programs. Following damage, Drosophila imaginal discs, the larval precursors of adult structures, can regenerate missing portions by localized proliferation of damage-adjacent tissue. Using single-cell transcriptomics in regenerating wing discs, a comprehensive view of the transcriptome of regenerating discs was obtained and two regeneration-specific cell populations within the blastema, Blastema1 and Blastema2 were identified. Collectively, these cells upregulate multiple genes encoding secreted proteins that promote regeneration including Pvf1, upd3, asperous, Mmp1, and the maturation delaying factor Ilp8. Expression of the transcription factor Ets21C is restricted to this regenerative secretory zone; it is not expressed in undamaged discs. Ets21C expression is activated by the JNK/AP-1 pathway, and it can function in a type 1 coherent feedforward loop with AP-1 to sustain expression of downstream genes. Without Ets21C function, the blastema cells fail to maintain the expression of a number of genes, which leads to premature differentiation and severely compromised regeneration. As Ets21C is dispensable for normal development, these observations indicate that Ets21C orchestrates a regeneration-specific gene regulatory network. This study has also identified cells resembling both Blastema1 and Blastema2 in scribble tumorous discs. They express the Ets21C-dependent gene regulatory network, and eliminating Ets21C function reduces tumorous growth. Thus, mechanisms that function during regeneration can be co-opted by tumors to promote aberrant growth.
Gass, M. M., Borkowsky, S., Lotz, M. L., Siwek, R., Schroter, R., Nedvetsky, P., Luschnig, S., Rohlmann, A., Missler, M. and Krahn, M. P. (2022). PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes. Cell Mol Life Sci 79(5): 248. PubMed ID: 35437696
Summary:
Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. This study demonstrates that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development. 		Deliu, L. P., Turingan, M., Jadir, D., Lee, B., Ghosh, A. and Grewal, S. S. (2022). Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development. PLoS Genet 18(9): e1010371. PubMed ID: 36048889
Summary:
The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. This study explored this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. This developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, this study showed that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. These effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development.
Ayukawa, T., Akiyama, M., Hozumi, Y., Ishimoto, K., Sasaki, J., Senoo, H., Sasaki, T. and Yamazaki, M. (2022). Tissue flow regulates planar cell polarity independently of the Frizzled core pathway. Cell Rep 40(12): 111388. PubMed ID: 36130497
Summary:
Planar cell polarity (PCP) regulates the orientation of external structures. A core group of proteins that includes Frizzled forms the heart of the PCP regulatory system. Other PCP mechanisms that are independent of the core group likely exist, but their underlying mechanisms are elusive. This study shows that tissue flow is a mechanism governing core group-independent PCP on the Drosophila notum. Loss of core group function only slightly affects bristle orientation in the adult central notum. This near-normal PCP results from tissue flow-mediated rescue of random bristle orientation during the pupal stage. Manipulation studies suggest that tissue flow can orient bristles in the opposite direction to the flow. This process is independent of the core group and implies that the apical extracellular matrix functions like a "comb" to align bristles. These results reveal the significance of cooperation between tissue dynamics and extracellular substances in PCP establishment. 		Stephenson, H. N., Streeck, R., Grublinger, F., Goosmann, C. and Herzig, A. (2022). Hemocytes are critical for Drosophila melanogaster post-embryonic development, independent of control of the microbiota. Development. PubMed ID: 36093870
Summary:
Proven roles for hemocytes (blood cells) have expanded beyond the control of infections in Drosophila. Despite this, the critical role of hemocytes in post-embryonic development has long thought to be limited to control of microorganisms during metamorphosis. This has previously been shown by rescue of adult development in hemocyte-ablation models under germ-free conditions. This study shows that hemocytes have a critical role in post-embryonic development beyond their ability to control the microbiota. Using a newly generated, strong hemocyte-specific driver line for the GAL4/UAS system, it was shown that specific ablation of hemocytes is early pupal lethal, even under axenic conditions. Genetic rescue experiments prove that this is a hemocyte-specific phenomena. RNA-seq data suggests that dysregulation of the midgut is a prominent consequence of hemocyte ablation in larval stages, resulting in reduced gut lengths. Dissection suggests that multiple processes may be affected during metamorphosis. It is believed that this novel role for hemocytes during metamorphosis is a major finding for the field.

Tuesday, November 8th - Gonads

Osswald, M., Barros-Carvalho, A., Carmo, A. M., Loyer, N., Gracio, P. C., Sunkel, C. E., Homem, C. C. F., Januschke, J. and Morais-de-Sa, E. (2022). aPKC regulates apical constriction to prevent tissue rupture in the Drosophila follicular epithelium. Curr Biol. PubMed ID: 36113470
Summary:
Apical-basal polarity is an essential epithelial trait controlled by the evolutionarily conserved PAR-aPKC polarity network. Dysregulation of polarity proteins disrupts tissue organization during development and in disease, but the underlying mechanisms are unclear due to the broad implications of polarity loss. This study uncovered how Drosophila aPKC maintains epithelial architecture by directly observing tissue disorganization after fast optogenetic inactivation in living adult flies and ovaries cultured ex vivo. Fast aPKC perturbation in the proliferative follicular epithelium produces large epithelial gaps that result from increased apical constriction, rather than loss of apical-basal polarity. Accordingly, it is possible to modulate the incidence of epithelial gaps by increasing and decreasing actomyosin-driven contractility. The origin of these large epithelial gaps were traced to tissue rupture next to dividing cells. Live imaging shows that aPKC perturbation induces apical constriction in non-mitotic cells within minutes, producing pulling forces that ultimately detach dividing and neighboring cells. It was further demonstrated that epithelial rupture requires a global increase of apical constriction, as it is prevented by the presence of non-constricting cells. Conversely, a global induction of apical tension through light-induced recruitment of RhoGEF2 to the apical side is sufficient to produce tissue rupture. Hence, this work reveals that the roles of aPKC in polarity and actomyosin regulation are separable and provides the first in vivo evidence that excessive tissue stress can break the epithelial barrier during proliferation. 		Jia, D., Jevitt, A., Huang, Y. C., Ramos, B. and Deng, W. M. (2022). Developmental regulation of epithelial cell cuboidal-to-squamous transition in Drosophila follicle cells. Dev Biol. PubMed ID: 36100084
Summary:
Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs), are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in twelve hours during midoogenesis. Previously, it was reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. This study reports that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for the SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study single-cell RNA sequencing data was used to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened.
Diegmiller, R., Nunley, H., Shvartsman, S. Y. and Imran Alsous, J. (2022). Quantitative models for building and growing fated small cell networks. Interface Focus 12(4): 20210082. PubMed ID: 35865502
Summary:
Small cell clusters exhibit numerous phenomena typically associated with complex systems, such as division of labour and programmed cell death. A conserved class of such clusters occurs during oogenesis in the form of germline cysts that give rise to oocytes. Germline cysts form through cell divisions with incomplete cytokinesis, leaving cells intimately connected through intercellular bridges that facilitate cyst generation, cell fate determination and collective growth dynamics. Using the well-characterized Drosophila melanogaster female germline cyst as a foundation, this study presents mathematical models rooted in the dynamics of cell cycle proteins and their interactions to explain the generation of germline cell lineage trees (CLTs) and highlight the diversity of observed CLT sizes and topologies across species. Competing models of symmetry breaking in CLTs were analyzed to rationalize the observed dynamics and robustness of oocyte fate specification, and highlight remaining gaps in knowledge. How CLT topology affects cell cycle dynamics and synchronization were analyzed and mechanisms of intercellular coupling are highlighted that underlie the observed collective growth patterns during oogenesis. Throughout, similarities across organisms are described that warrant further investigation and comments are made on the extent to which experimental and theoretical findings made in model systems extend to other species. 		Valentino, M., Ortega, B. M., Ulrich, B., Doyle, D. A., Farnum, E. D., Joiner, D. A., Gavis, E. R. and Niepielko, M. G. (2022). Computational modeling offers new insight into Drosophila germ granule development. Biophys J 121(8): 1465-1482. PubMed ID: 35288123
Summary:
The packaging of specific mRNAs into ribonucleoprotein granules called germ granules is required for germline proliferation and maintenance. During Drosophila germ granule development, mRNAs such as nanos (nos) and polar granule component (pgc) localize to germ granules through a stochastic seeding and self-recruitment process that generates homotypic clusters: aggregates containing multiple copies of a specific transcript. Germ granules vary in mRNA composition with respect to the different transcripts that they contain and their quantity. However, what influences germ granule mRNA composition during development is unclear. To gain insight into how germ granule mRNA heterogeneity arises, this study created a computational model that simulates granule development. Although the model includes known mechanisms that were converted into mathematical representations, additional unreported mechanisms proved to be essential for modeling germ granule formation. The model was validated by predicting defects caused by changes in mRNA and protein abundance. Broader application of the model was demonstrated by quantifying nos and pgc localization efficacies and the contribution that an element within the nos 3' untranslated region has on clustering. For the first time, a mathematical representation of Drosophila germ granule formation is described, offering quantitative insight into how mRNA compositions arise while providing a new tool for guiding future studies.
Zhao, T., Xiao, Y., Huang, B., Ran, M. J., Duan, X., Wang, Y. F., Lu, Y. and Yu, X. Q. (2022). tA dual role of lola in Drosophila ovary development: regulating stem cell niche establishment and repressing apoptosis. Cell Death Dis 13(9): 756. PubMed ID: 36056003
Summary:
In Drosophila ovary, niche is composed of somatic cells, including terminal filament cells (TFCs), cap cells (CCs) and escort cells (ECs), which provide extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Niche establishment begins in larval stages when terminal filaments (TFs) are formed, but the underlying mechanism for the development of TFs remains largely unknown. This study reports that transcription factor longitudinals lacking (Lola) is essential for ovary morphogenesis. Lola protein was expressed abundantly in TFCs and CCs, although also in other cells, and lola was required for the establishment of niche during larval stage. Importantly, it was found that knockdown expression of lola induced apoptosis in adult ovary, and that lola affected adult ovary morphogenesis by suppressing expression of Regulator of cullins 1b (Roc1b), an apoptosis-related gene that regulates caspase activation during spermatogenesis. These findings significantly expand understanding of the mechanisms controlling niche establishment and adult oogenesis in Drosophila. 		Anllo, L. and DiNardo, S. (2022). Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche. Dev Cell 57(8): 1009-1023.e1005. PubMed ID: 35390292
Summary:
Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis, recent work showed that pro-niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which was found to be also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. The function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. This work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

Monday, November 7th - Physiology and Metabolism

Singh, V. J., Potdar, S. and Sheeba, V. (2022). Effects of Food Availability Cycles on Phase and Period of Activity-rest Rhythm in Drosophila melanogaster. J Biol Rhythms 37(5): 528-544. PubMed ID: 35983646
Summary:
Foraging and feeding are indispensable for survival and their timing depends not only on the metabolic state of the animal but also on the availability of food resources in their environment. Since both these aspects are subject to change over time, these behaviors exhibit rhythmicity in occurrence. As the locomotor activity of an organism is related to its disposition to acquire food, and peak feeding in fruit flies has been shown to occur at a particular time of the day, it was asked if cyclic food availability can entrain their rhythmic activity. By subjecting flies to cyclic food availability, that is, feeding-starvation (FS) cycles, food cues were provided contrasting to the preferred activity times, and whether this imposed cycling in food availability could entrain the activity-rest rhythm was studied. Phase control, which is a property integral to entrainment, was not achieved despite increasing starvation duration of FS cycles (FS 12:12, FS 10:14, and FS 8:16). It was also found that flies subjected to T21 and T26 FS zeitgeber cycles were unable to match period of the activity rhythm to short or long T-cycles. Taken together, these results show that external food availability cycles do not entrain the activity-rest rhythm of fruit flies. However, it was found that starvation-induced hyperactivity causes masking which results in phase changes. In addition, T-cycle experiments resulted in minor period changes during FS treatment. These findings highlight that food cyclicity by itself may not be a potent zeitgeber but may act in unison with other abiotic factors like light and temperature to help flies time their activity appropriately. 		Casey, A. K., Gray, H. F., Chimalapati, S., Hernandez, G., Moehlman, A. T., Stewart, N., Fields, H. A., Gulen, B., Servage, K. A., Stefanius, K., Blevins, A., Evers, B. M., Kramer, H. and Orth, K. (2022). Fic-mediated AMPylation tempers the unfolded protein response during physiological stress. Proc Natl Acad Sci U S A 119(32): e2208317119. PubMed ID: 35914137
Summary:
The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, it was discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, a conditional null allele of Fic was induced in mice, and the effect of Fic loss on the exocrine pancreas was characterized. Compared to controls, Fic(-/-) mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic(-/-) flies and Fic(-/-) mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, it is proposed that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.
Werner, E., Gokhale, A., Ackert, M., Xu, C., Wen, Z., Roberts, A. M., Roberts, B. R., Vrailas-Mortimer, A., Crocker, A. and Faundez, V. (2022). The mitochondrial RNA granule modulates manganese-dependent cell toxicity. Mol Biol Cell 33(12): ar108. PubMed ID: 35921164
Summary:
Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and nonadaptive cellular processes targeted by manganese remains mostly unexplored. This study looked at chanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinson's gene PARK2. It was found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule composition and function, resulting in disruption of mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. It is concluded that impairment of the mitochondrial RNA granule function is a protective mechanism for acute manganese toxicity. 		De Lazzari, F., Agostini, F., Doni, D., Malacrida, S., Zordan, M. A., Costantini, P., Bubacco, L., Sandrelli, F. and Bisaglia, M. (2022). DJ-1 and SOD1 Act Independently in the Protection against Anoxia in Drosophila melanogaster. Antioxidants (Basel) 11(8). PubMed ID: 36009245
Summary:
Redox homeostasis is a vital process the maintenance of which is assured by the presence of numerous antioxidant small molecules and enzymes and the alteration of which is involved in many pathologies, including several neurodegenerative disorders. Among the different enzymes involved in the antioxidant response, SOD1 and DJ-1 have both been associated with the pathogenesis of amyotrophic lateral sclerosis and Parkinson's disease, suggesting a possible interplay in their mechanism of action. Copper deficiency in the SOD1-active site has been proposed as a central determinant in SOD1-related neurodegeneration. SOD1 maturation mainly relies on the presence of the protein copper chaperone for SOD1 (CCS), but a CCS-independent alternative pathway also exists and functions under anaerobic conditions. To explore the possible involvement of DJ-1 in such a pathway in vivo, Drosophila melanogaster was exposed to anoxia and the effect was evaluated of DJ-1 on fly survival and SOD1 levels, in the presence or absence of CCS. Loss of DJ-1 negatively affects the fly response to the anoxic treatment, but the data indicate that the protective activity of DJ-1 is independent of SOD1 in Drosophila, indicating that the two proteins may act in different pathways.
Shang, L., Aughey, E., Kim, H., Heden, T. D., Wang, L., Najt, C. P., Esch, N., Brunko, S., Abrahante, J. E., Macchietto, M., Mashek, M. T., Fairbanks, T., Promislow, D. E. L., Neufeld, T. P. and Mashek, D. G. (2022). Systemic lipolysis promotes physiological fitness in Drosophila melanogaster. Aging (Albany NY) 14(16): 6481-6506. PubMed ID: 36044277
Summary:
Since interventions such as caloric restriction or fasting robustly promote lipid catabolism and improve aging-related phenotypical markers, this study investigated the direct effect of increased lipid catabolism via overexpression of bmm (brummer, the major triglyceride hydrolase in Drosophila, on lifespan and physiological fitness. Comprehensive characterization was carried out using RNA-seq, lipidomics and metabolomics analysis. Global overexpression of bmm strongly promoted numerous markers of physiological fitness, including increased female fecundity, fertility maintenance, preserved locomotion activity, increased mitochondrial biogenesis and oxidative metabolism. Increased bmm robustly upregulated the heat shock protein 70 (Hsp70) family of proteins, which equipped the flies with higher resistance to heat, cold, and ER stress via improved proteostasis. Despite improved physiological fitness, bmm overexpression did not extend lifespan. Taken together, these data show that bmm overexpression has broad beneficial effects on physiological fitness, but these effects did not impact lifespan. 		Strilbytska, O., Semaniuk, U., Bubalo, V., Storey, K. B. and Lushchak, O. (2022). Dietary Choice Reshapes Metabolism in Drosophila by Affecting Consumption of Macronutrients. Biomolecules 12(9). PubMed ID: 36139040
Summary:
The precise regulation of metabolism and feeding behavior is important for preventing the development of metabolic diseases. This study examined the effects on Drosophila metabolism of dietary choice. These changes are predicted to be dependent on both the quantity and quality of the chosen diet. Using a geometric framework for both no-choice and two-choice conditions, it was found that feeding decisions led to higher glucose and trehalose levels but lower triglycerides pools. The feeding regimens had similar strategies for macronutrient balancing, and both maximized hemolymph glucose and glycogen content under low protein intake. In addition, the flies showed significant differences in the way they regulated trehalose and triglyceride levels in response to carbohydrate and protein consumption between choice and no-choice nutrition. Under choice conditions, trehalose and triglyceride levels were maximized at the lowest protein and carbohydrate consumption. Thus, it is suggested that these changes in carbohydrate and lipid metabolism are caused by differences in the macronutrients consumed by flies. Food choice elicits rapid metabolic changes to maintain energy homeostasis. These results contribute to understanding of how metabolism is regulated by the revealed nutrient variation in response to food decisions.

Friday, November 4th - Disease Models

Thackray, A. M., Lam, B., McNulty, E. E., Nalls, A. V., Mathiason, C. K., Magadi, S. S., Jackson, W. S., Andreoletti, O., Marrero-Winkens, C., Schatzl, H. and Bujdoso, R. (2022). Clearance of variant Creutzfeldt-Jakob disease prions in vivo by the Hsp70 disaggregase system. Brain 145(9): 3236-3249. PubMed ID: 35446941
Summary:
The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. This study tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so prion protein (PrP) transgenic Drosophila were used that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was downregulated in prion-exposed PrP Drosophila. RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas overexpression mitigated toxicity. Strikingly, prion seeding activity in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 overexpression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease. 		Ayajuddin, M., Phom, L., Koza, Z., Modi, P., Das, A., Chaurasia, R., Thepa, A., Jamir, N., Neikha, K. and Yenisetti, S. C. (2022). Adult health and transition stage-specific rotenone-mediated Drosophila model of Parkinson's disease: Impact on late-onset neurodegenerative disease models. Front Mol Neurosci 15: 896183. PubMed ID: 36017079
Summary:
Parkinson's disease (PD) affects almost 1% of the population worldwide over the age of 50 years. Exposure to environmental toxins like paraquat and rotenone is a risk factor for sporadic PD which constitutes 95% of total cases. Herbicide rotenone has been shown to cause Parkinsonian symptoms in multiple animal models. Drosophila is an excellent model organism for studying neurodegenerative diseases (NDD) including PD. The aging process is characterized by differential expression of genes during different life stages. Hence it is necessary to develop life-stage-matched animal models for late-onset human disease(s) such as PD. Such animal models are critical for understanding the pathophysiology of age-related disease progression and important to understand if a genotropic drug/nutraceutical can be effective during late stages. With this idea, an adult life stage-specific (health and transition phase, during which late-onset NDDs such as PD sets in) rotenone-mediated Drosophila model of idiopathic PD was developed. Drosophila is susceptible to rotenone in dose-time dependent manner. Rotenone-mediated fly model of sporadic PD exhibits mobility defects (independent of mortality), inhibited mitochondrial complex I activity, dopaminergic (DAergic) neuronal dysfunction (no loss of DAergic neuronal number; however, reduction in rate-limiting enzyme tyrosine hydroxylase (TH) synthesis), and alteration in levels of dopamine (DA) and its metabolites; 3,4-Dihydroxyphenylacetic acid (DOPAC) and Homovanilic acid (HVA) in brain-specific fashion. These PD-linked behaviors and brain-specific phenotypes denote the robustness of the present fly model of PD. This novel model will be of great help to decipher life stage-specific genetic targets of small molecule mediated DAergic neuroprotection; understanding of which is critical for formulating therapeutic strategies for PD.
Tonoki, A., Nagai, S., Yu, Z., Yue, T., Lyu, S., Hou, X., Onuki, K., Yabana, K., Takahashi, H. and Itoh, M. (2022). Nitric oxide-soluble guanylyl cyclase pathway as a contributor to age-related memory impairment in Drosophila. Aging Cell 21(9): e13691. PubMed ID: 35963012
Summary:
Age-related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age-dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. A list of candidate genes that act as AMI regulators was generated by performing a comprehensive analysis of RNA sequencing data from young and aged fly heads and genome-wide RNAi screening data to identify memory-regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. The guanylyl cyclase β-subunit at 100B (gycβ) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycβ in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate-term memory (ITM) without apparent effects on memory acquisition. This study also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age-related enhancement of the NO-sGC pathway causes memory impairment. 		Al-Amri, A. H., Armstrong, P., Amici, M., Ligneul, ..., Ali, M., Inglehearn, C. F. and Clapcote, S. J. (2022). PDZD8 Disruption Causes Cognitive Impairment in Humans, Mice, and Fruit Flies. Biol Psychiatry 92(4): 323-334. PubMed ID: 35227461
Summary:
The discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability. Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene's function in cognition, Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing. Homozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum-anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits. These data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.
Sung, H. and Lloyd, T. E. (2022). Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD. Traffic 23(9): 430-441. PubMed ID: 35908282
Summary:
A GGGGCC (G(4) C(2)) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. This study performed live imaging of Drosophila motor neurons expressing expanded G(4) C(2) repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G(4) C(2) repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G(4) C(2) repeats. Thus,these data indicate that expanded G(4) C(2) repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD. 		Zhang, S., Zhu, Y., Lu, J., Liu, Z., Lobato, A. G., Zeng, W., Liu, J., Qiang, J., Zeng, S., Zhang, Y., Liu, C., Liu, J., He, Z., Zhai, R. G. and Li, D. (2022). Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology. Elife 11. PubMed ID: 36048712
Summary:
Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. This study, shows that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. This work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

Thursday, November 3rd - Stem Cells

Sreejith, P., Malik, S., Kim, C. and Biteau, B. (2022). Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestine. PLoS Genet 18(9): e1010385. PubMed ID: 36070313
Summary:
Stem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. This report shows that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. Imp was demonstrated to be required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically,Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. Both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, these data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis. 		Zhang, C., Jin, Y., Marchetti, M., Lewis, M. R., Hammouda, O. T. and Edgar, B. A. (2022). EGFR signaling activates intestinal stem cells by promoting mitochondrial biogenesis and beta-oxidation. Curr Biol 32(17): 3704-3719. PubMed ID: 35896119
Summary:
EGFR-RAS-ERK signaling promotes growth and proliferation in many cell types, and genetic hyperactivation of RAS-ERK signaling drives many cancers. Yet, despite intensive study of upstream components in EGFR signal transduction, the identities and functions of downstream effectors in the pathway are poorly understood. In Drosophila intestinal stem cells (ISCs), the transcriptional repressor Capicua (Cic) and its targets, the ETS-type transcriptional activators Pointed (pnt) and Ets21C, are essential downstream effectors of mitogenic EGFR signaling. This study shows that these factors promote EGFR-dependent metabolic changes that increase ISC mass, mitochondrial growth, and mitochondrial activity. Gene target analysis using RNA and DamID sequencing revealed that Pnt and Ets21C directly upregulate not only DNA replication and cell cycle genes but also genes for oxidative phosphorylation, the TCA cycle, and fatty acid beta-oxidation. Metabolite analysis substantiated these metabolic functions. The mitochondrial transcription factor B2 (mtTFB2), a direct target of Pnt, was required and partially sufficient for EGFR-driven ISC growth, mitochondrial biogenesis, and proliferation. MEK-dependent EGF signaling stimulated mitochondrial biogenesis in human RPE-1 cells, indicating the conservation of these metabolic effects. This work illustrates how EGFR signaling alters metabolism to coordinately activate cell growth and cell division.
Lee, S. H., Hwang, D., Goo, T. W. and Yun, E. Y. (2022). Prediction of intestinal stem cell regulatory genes from Drosophila gut damage model created using multiple inducers: Differential gene expression-based protein-protein interaction network analysis. Dev Comp Immunol 138: 104539. PubMed ID: 36087786
Summary:
Intestinal tissue functions in innate immunity to prevent the entry of harmful substances, and to maintain homeostasis through the constant proliferation of intestinal stem cells (ISC). To understand the mechanisms which regulate ISC in response to gut damage, this study identified 81 differentially expressed genes (DEGs) through RNA-seq analysis after oral administration of three intestinal-damaging substances to Drosophila melanogaster. Through protein-protein interaction (PPI) and functional annotation studies, the top 22 DEGs ordered by the number of nodes in the PPI network were analyzed in relation to cell development. Through network topology analysis, 12 essential seed genes were identified. From this it can be confirmed that p53, RpL17, Fmr1, Stat92E, CG31343, Cnot4, CG9281, CG8184, Evi5, and to were essential for ISC proliferation during gut damage using knockdown RNAi Drosophila. This study presents a method for identifying candidate genes relating to intestinal damage that has scope for furthering understanding of gut disease. 		Senos Demarco, R., Stack, B. J., Tang, A. M., Voog, J., Sandall, S. L., Southall, T. D., Brand, A. H. and Jones, D. L. (2022). Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila. Cell Rep 39(3): 110679. PubMed ID: 35443165
Summary:
Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. This study shows that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.
Ren, X., Zhao, H., Shi, L., Li, Z., Kong, R., Ma, R., Jia, L., Lu, S., Wang, J. H., Dong, M. Q., Wang, Y. and Li, Z. (2022). Phosphorylation of Yun is required for stem cell proliferation and tumorigenesis. Cell Prolif 55(5): e13230. PubMed ID: 35437864
Summary:
Stem cells maintain adult tissue homeostasis under physiological conditions. Uncontrolled stem cell proliferation will lead to tumorigenesis. How stem cell proliferation is precisely controlled is still not fully understood. Phosphorylation of Yun is essential for ISC proliferation. Yun is essential for the proliferation of normal and transformed intestinal stem cells. Mass spectrometry and biochemical data suggest that Yun can be phosphorylated at multiple residues in vivo. Interestingly, it was shown that the phosphorylation among these residues is likely interdependent. Furthermore, phosphorylation of each residue in Yun is important for its function in ISC proliferation regulation. Thus, this study unveils the important role of post-translational modification of Yun in stem cell proliferation. 		Zhao, H., Li, Z., Kong, R., Shi, L., Ma, R., Ren, X. and Li, Z. (2022). Novel intrinsic factor Yun maintains female germline stem cell fate through Thickveins. Stem Cell Reports 17(9): 1914-1923. PubMed ID: 35985332
Summary:
Germline stem cells (GSCs) are critical for the reproduction of an organism. The self-renewal and differentiation of GSCs must be tightly controlled to avoid uncontrolled stem cell proliferation or premature stem cell differentiation. However, how the self-renewal and differentiation of GSCs are properly controlled is not fully understood. This study finds that the novel intrinsic factor Yun is required for female GSC maintenance in Drosophila. GSCs undergo precocious differentiation due to de-repression of differentiation factor Bam by defective BMP/Dpp signaling in the absence of yun. Mechanistically, Yun associates with and stabilizes Thickveins (Tkv), the type I receptor of Dpp/BMP signaling. Finally, ectopic expression of a constitutively active Tkv (Tkv(QD)) completely suppresses GSC loss caused by yun depletion. Collectively, these data demonstrate that Yun functions through Tkv to maintain GSC fate. These results provide new insight into the regulatory mechanisms of how stem cell maintenance is properly controlled.

Wednesday November 2nd - Larval and Adult Neural Development and Function

Zhang, X., Sabandal, J. M., Tsaprailis, G. and Davis, R. L. (2022). Active forgetting requires Sickie function in a dedicated dopamine circuit in Drosophila. Proc Natl Acad Sci U S A 119(38): e2204229119. PubMed ID: 36095217
Summary:
Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. This study shows that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca(2+) influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, this study showed that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. These results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release. 		Valentino, P. and Erclik, T. (2022). Spalt and Disco Define the Dorsal-Ventral Neuroepithelial Compartments of the Developing Drosophila Medulla. Genetics. PubMed ID: 36135799
Summary:
Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center (OPC) crescent are spatially patterned by the non-overlapping expression of three transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the OPC and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the OPC also grow as distinct compartments, together with the fact that a subset of neuronal types are generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the OPC along the dorsal-ventral (D-V) axis. This study identified the spalt (salm and salr) and disco (discoand disco-r) genes as the D-V patterning transcription factors of the OPC. Spalt and Disco are differentially expressed in the dorsal and ventral OPC from the embryo through to the third instar larva, where they cross-repress each other to form a sharp D-V boundary. hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral OPC and its neuronal progeny. It was further demonstrated that this D-V patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third OPC patterning axis that may act to further diversify medulla fates. .
Villar, M. E., Pavao-Delgado, M., Amigo, M., Jacob, P. F., Merabet, N., Pinot, A., Perry, S. A., Waddell, S. and Perisse, E. (2022). Differential coding of absolute and relative aversive value in the Drosophila brain. Curr Biol. PubMed ID: 36103878
Summary:
Animals use prior experience to assign absolute (good or bad) and relative (better or worse) value to new experience. These learned values guide appropriate later decision making. Even though understanding of how the valuation system computes absolute value is relatively advanced, the mechanistic underpinnings of relative valuation are unclear. Yhid study uncovered mechanisms of absolute and relative aversive valuation in Drosophila. Three types of punishment-sensitive dopaminergic neurons (DANs) respond differently to electric shock intensity. During learning, these punishment-sensitive DANs drive intensity-scaled plasticity at their respective mushroom body output neuron (MBON) connections to code absolute aversive value. In contrast, by comparing the absolute value of current and previous aversive experiences, the MBON-DAN network can code relative aversive value by using specific punishment-sensitive DANs and recruiting a specific subtype of reward-coding DANs. Behavioral and physiological experiments revealed that a specific subtype of reward-coding DAN assigns a "better than" value to the lesser of the two aversive experiences. This study therefore highlights how appetitive-aversive system interactions within the MB network can code and compare sequential aversive experiences to learn relative aversive value 		Ryu, T. H., Subramanian, M., Yeom, E. and Yu, K. (2022). The prominin-like Gene Expressed in a Subset of Dopaminergic Neurons Regulates Locomotion in Drosophila. Mol Cells 45(9): 640-648. PubMed ID: 35993164
Summary:
CD133, also known as prominin-1, was first identified as a biomarker of mammalian cancer and neural stem cells. Previous studies have shown that the prominin-like (promL) gene, an orthologue of mammalian CD133 in Drosophila, plays a role in glucose and lipid metabolism, body growth, and longevity. Because locomotion is required for food sourcing and ultimately the regulation of metabolism, this study examined the function of promL in Drosophila locomotion. Both promL mutants and pan-neuronal promL inhibition flies displayed reduced spontaneous locomotor activity. As dopamine is known to modulate locomotion, the effects of promL inhibition on the dopamine concentration and mRNA expression levels of tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc), the enzymes that are responsible for dopamine biosynthesis, were examined in the heads of flies. Compared with those in control flies, the levels of dopamine and the mRNAs encoding TH and Ddc were lower in promL mutant and pan-neuronal promL inhibition flies. In addition, an immunostaining analysis revealed that, compared with control flies, promL mutant and pan-neuronal promL inhibition flies had lower levels of the TH protein in protocerebral anterior medial (PAM) neurons, a subset of dopaminergic neurons. Inhibition of promL in these PAM neurons reduced the locomotor activity of the flies. Overall, these findings indicate that promL expressed in PAM dopaminergic neurons regulates locomotion by controlling dopamine synthesis in Drosophila.
Sorkac, A., Savva, Y. A., Savaş, D., Talay, M. and Barnea, G. (2022). Circuit analysis reveals a neural pathway for light avoidance in Drosophila larvae. Nat Commun 13(1): 5274. PubMed ID: 36071059
Summary:
Understanding how neural circuits underlie behaviour is challenging even in the connectome era because it requires a combination of anatomical and functional analyses. This is exemplified in the circuit underlying the light avoidance behaviour displayed by Drosophila melanogaster larvae. While this behaviour is robust and the nervous system relatively simple, the circuit is only partially delineated with some contradictions among studies. This study devised trans-Tango MkII, an offshoot of the transsynaptic circuit tracing tool trans-Tango, and implement it in anatomical tracing together with functional analysis. Neuronal inhibition was used to test necessity of particular neuronal types in light avoidance and selective neuronal activation to examine sufficiency in rescuing light avoidance deficiencies exhibited by photoreceptor mutants. These studies reveal a four-order circuit for light avoidance connecting the light-detecting photoreceptors with a pair of neuroendocrine cells via two types of clock neurons. This approach can be readily expanded to studying other circuits. 		Snell, N. J., Fisher, J. D., Hartmann, G. G., Zolyomi, B., Talay, M. and Barnea, G. (2022). Complex representation of taste quality by second-order gustatory neurons in Drosophila. Curr Biol 32(17): 3758-3772.e3754. PubMed ID: 35973432
Summary:
Sweet and bitter compounds excite different sensory cells and drive opposing behaviors. However, it remains unclear how sweet and bitter tastes are represented by the neural circuits linking sensation to behavior. To investigate this question in Drosophila, this study devised trans-Tango(activity), a strategy for calcium imaging of second-order gustatory projection neurons based on trans-Tango, a genetic transsynaptic tracing technique. Spatial overlap was found between the projection neuron populations activated by sweet and bitter tastants. The spatial representation of bitter tastants in the projection neurons was consistent, while that of sweet tastants was heterogeneous. Furthermore, it wads discovered that bitter tastants evoke responses in the gustatory receptor neurons and projection neurons upon both stimulus onset and offset and that bitter offset and sweet onset excite overlapping second-order projections. These findings demonstrate an unexpected complexity in the representation of sweet and bitter tastants by second-order neurons of the gustatory circuit.

Tuesday, November 1st - Signal Transduction

Zhang, X., Sabandal, J. M., Tsaprailis, G. and Davis, R. L. (2022). Active forgetting requires Sickie function in a dedicated dopamine circuit in Drosophila. Proc Natl Acad Sci U S A 119(38): e2204229119. PubMed ID: 36095217
Summary:
Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. This study shows that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca(2+) influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, this study showed that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. These results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release. 		Valentino, P. and Erclik, T. (2022). Spalt and Disco Define the Dorsal-Ventral Neuroepithelial Compartments of the Developing Drosophila Medulla. Genetics. PubMed ID: 36135799
Summary:
Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center (OPC) crescent are spatially patterned by the non-overlapping expression of three transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the OPC and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the OPC also grow as distinct compartments, together with the fact that a subset of neuronal types are generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the OPC along the dorsal-ventral (D-V) axis. This study identified the spalt (salm and salr) and disco (disco and disco-r) genes as the D-V patterning transcription factors of the OPC. Spalt and Disco are differentially expressed in the dorsal and ventral OPC from the embryo through to the third instar larva, where they cross-repress each other to form a sharp D-V boundary. hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral OPC and its neuronal progeny. It was further demonstrated that this D-V patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third OPC patterning axis that may act to further diversify medulla fates. .
Villar, M. E., Pavao-Delgado, M., Amigo, M., Jacob, P. F., Merabet, N., Pinot, A., Perry, S. A., Waddell, S. and Perisse, E. (2022). Differential coding of absolute and relative aversive value in the Drosophila brain. Curr Biol. PubMed ID: 36103878
Summary:
Animals use prior experience to assign absolute (good or bad) and relative (better or worse) value to new experience. These learned values guide appropriate later decision making. Even though understanding of how the valuation system computes absolute value is relatively advanced, the mechanistic underpinnings of relative valuation are unclear. Yhid study uncovered mechanisms of absolute and relative aversive valuation in Drosophila. Three types of punishment-sensitive dopaminergic neurons (DANs) respond differently to electric shock intensity. During learning, these punishment-sensitive DANs drive intensity-scaled plasticity at their respective mushroom body output neuron (MBON) connections to code absolute aversive value. In contrast, by comparing the absolute value of current and previous aversive experiences, the MBON-DAN network can code relative aversive value by using specific punishment-sensitive DANs and recruiting a specific subtype of reward-coding DANs. Behavioral and physiological experiments revealed that a specific subtype of reward-coding DAN assigns a "better than" value to the lesser of the two aversive experiences. This study therefore highlights how appetitive-aversive system interactions within the MB network can code and compare sequential aversive experiences to learn relative aversive value 		Ryu, T. H., Subramanian, M., Yeom, E. and Yu, K. (2022). The prominin-like Gene Expressed in a Subset of Dopaminergic Neurons Regulates Locomotion in Drosophila. Mol Cells 45(9): 640-648. PubMed ID: 35993164
Summary:
CD133, also known as prominin-1, was first identified as a biomarker of mammalian cancer and neural stem cells. Previous studies have shown that the prominin-like (promL) gene, an orthologue of mammalian CD133 in Drosophila, plays a role in glucose and lipid metabolism, body growth, and longevity. Because locomotion is required for food sourcing and ultimately the regulation of metabolism, this study examined the function of promL in Drosophila locomotion. Both promL mutants and pan-neuronal promL inhibition flies displayed reduced spontaneous locomotor activity. As dopamine is known to modulate locomotion, the effects of promL inhibition on the dopamine concentration and mRNA expression levels of tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc), the enzymes that are responsible for dopamine biosynthesis, were examined in the heads of flies. Compared with those in control flies, the levels of dopamine and the mRNAs encoding TH and Ddc were lower in promL mutant and pan-neuronal promL inhibition flies. In addition, an immunostaining analysis revealed that, compared with control flies, promL mutant and pan-neuronal promL inhibition flies had lower levels of the TH protein in protocerebral anterior medial (PAM) neurons, a subset of dopaminergic neurons. Inhibition of promL in these PAM neurons reduced the locomotor activity of the flies. Overall, these findings indicate that promL expressed in PAM dopaminergic neurons regulates locomotion by controlling dopamine synthesis in Drosophila.
Sorkac, A., Savva, Y. A., Savaş, D., Talay, M. and Barnea, G. (2022). Circuit analysis reveals a neural pathway for light avoidance in Drosophila larvae. Nat Commun 13(1): 5274. PubMed ID: 36071059
Summary:
Understanding how neural circuits underlie behaviour is challenging even in the connectome era because it requires a combination of anatomical and functional analyses. This is exemplified in the circuit underlying the light avoidance behaviour displayed by Drosophila melanogaster larvae. While this behaviour is robust and the nervous system relatively simple, the circuit is only partially delineated with some contradictions among studies. This study devised trans-Tango MkII, an offshoot of the transsynaptic circuit tracing tool trans-Tango, and implement it in anatomical tracing together with functional analysis. Neuronal inhibition was used to test necessity of particular neuronal types in light avoidance and selective neuronal activation to examine sufficiency in rescuing light avoidance deficiencies exhibited by photoreceptor mutants. These studies reveal a four-order circuit for light avoidance connecting the light-detecting photoreceptors with a pair of neuroendocrine cells via two types of clock neurons. This approach can be readily expanded to studying other circuits. 		Snell, N. J., Fisher, J. D., Hartmann, G. G., Zolyomi, B., Talay, M. and Barnea, G. (2022). Complex representation of taste quality by second-order gustatory neurons in Drosophila. Curr Biol 32(17): 3758-3772.e3754. PubMed ID: 35973432
Summary:
Sweet and bitter compounds excite different sensory cells and drive opposing behaviors. However, it remains unclear how sweet and bitter tastes are represented by the neural circuits linking sensation to behavior. To investigate this question in Drosophila, this study devised trans-Tango(activity), a strategy for calcium imaging of second-order gustatory projection neurons based on trans-Tango, a genetic transsynaptic tracing technique. Spatial overlap was found between the projection neuron populations activated by sweet and bitter tastants. The spatial representation of bitter tastants in the projection neurons was consistent, while that of sweet tastants was heterogeneous. Furthermore, it wads discovered that bitter tastants evoke responses in the gustatory receptor neurons and projection neurons upon both stimulus onset and offset and that bitter offset and sweet onset excite overlapping second-order projections. These findings demonstrate an unexpected complexity in the representation of sweet and bitter tastants by second-order neurons of the gustatory circuit.

Monday, November 1st - Signal transductionn

Sayeesh, P. M., Ikeya, T., Sugasawa, H., Watanabe, R., Mishima, M., Inomata, K. and Ito, Y. (2022). Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos. Biochem Biophys Res Commun 625: 87-93. PubMed ID: 35952612
Summary:
Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains NMR studies of Drk-CSH3 were performed. The resulting solution structure and the (15)N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk. 		Trivedi, S., Bhattacharya, M. and Starz-Gaiano, M. (2022). Mind bomb 2 promotes cell migration and epithelial structure by regulating adhesion complexes and the actin cytoskeleton. Dev Biol 491: 94-104. PubMed ID: 36067835
Summary:
Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, it was found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, components of Mib2 complexes were identified, includeing E-cadherin and α- and β-catenins, as well as actin regulators. Three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. It is concluded that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms.
Wassarman, D. R., Bankapalli, K., Pallanck, L. J. and Shokat, K. M. (2022). Tissue-restricted inhibition of mTOR using chemical genetics. Proc Natl Acad Sci U S A 119(38): e2204083119. PubMed ID: 36095197
Summary:
Mammalian target of rapamycin (mTOR: See Drosophila Tor) is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism, and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product rapamycin, are administered systemically and target multiple tissues simultaneously. This study developed a chemical-genetic system, termed selecTOR, that restricts the activity of a rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this rapamycin analog. Using this system, it was shown that selective mTOR inhibition can be achieved in Saccharomyces cerevisiae and human cells, and The utility of this system was developed in an intact metazoan model organism the tissues responsible for a rapamycin-induced developmental delay in Drosophila were identified. 		Wu, H., Zhu, N., Liu, J., Ma, J. and Jiao, R. (2022). Shaggy regulates tissue growth through Hippo pathway in Drosophila. Sci China Life Sci. PubMed ID: 36057002
Summary:
The evolutionarily conserved Hippo pathway coordinates cell proliferation, differentiation and apoptosis to regulate organ growth and tumorigenesis. Hippo signaling activity is tightly controlled by various upstream signals including growth factors and cell polarity, but the full extent to which the pathway is regulated during development remains to be resolved. This study reports the identification of Shaggy, the homolog of mammalian Gsk3β, as a novel regulator of the Hippo pathway in Drosophila. These results show that Shaggy promotes the expression of Hippo target genes in a manner that is dependent on its kinase activity. Loss of Shaggy leads to Yorkie inhibition and downregulation of Hippo pathway target genes. Mechanistically, Shaggy acts upstream of the Hippo pathway and negatively regulates the abundance of the FERM domain containing adaptor protein Expanded. These results reveal that Shaggy is functionally required for Crumbs/Slmb-mediated downregulation of Expanded in vivo, providing a potential molecular link between cellular architecture and the Hippo signaling pathway.
Nellas, I., Iyer, K. V., Iglesias-Artola, J. M., Pippel, M., Nadler, A., Eaton, S. and Dye, N. A. (2022). Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc. EMBO Rep: e202154025. PubMed ID: 36134875
Summary:
Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. This study used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, this work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development. 		Sharma, V., Sarkar, B., Mutsuddi, M. and Mukherjee, A. (2022).. Deltex modulates Dpp morphogen gradient formation and affects Dpp signaling in Drosophila. J Cell Sci 135(17). PubMed ID: 35950520
Summary:
Deltex (Dx) is a context-dependent regulator of Notch signaling that can act in a non-canonical fashion by facilitating the endocytosis of the Notch receptor. In an RNAi-based modifier screen of kinases and phosphatases, this study identified Thickveins (Tkv), the receptor of Decapentaplegic (Dpp), as one of the interactors of Dx. Dpp, a Drosophila homolog of TGF-β and bone morphogenetic proteins, acts as a morphogen to specify cell fate along the anterior-posterior axis of the wing. Tight regulation of Dpp signaling is thus indispensable for its proper functioning. This study presents Dx as a novel modulator of Dpp signaling. Evidence is shown for the very first time that dx genetically interacts with dpp and its pathway components. Immunocytochemical analysis revealed that Dx colocalizes with Dpp and its receptor Tkv in Drosophila third-instar larval tissues. Furthermore, Dx was also seen to modulate the expression of dpp and its target genes, and this modulation is attributed to the involvement of Dx in the endocytosis and trafficking of Dpp. This study thus presents a whole new avenue of Dpp signaling regulation via the cytoplasmic protein Dx.
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