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Thursday, June 30th, 2022 - Characterization of Genes and Protein

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Zafar, Z., Fatima, S., Bhatti, M. F., Shah, F. A., Saud, Z. and Butt, T. M. (2022). Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights. PLoS One 17(3): e0265896. PubMed ID: 35316281
Summary:
Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.
Pegoraro, M., Sayegh Rezek, E., Fishman, B. and Tauber, E. (2022). Nucleotide Variation in Drosophila cryptochrome Is Linked to Circadian Clock Function: An Association Analysis. Front Physiol 13: 781380. PubMed ID: 35250608
Summary:
Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila, cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. This study has generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, association mapping was used. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). A maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, the effect of B1/B2 polymorphism on circadian phase was verified. The transgenic flies also exhibited substantially different levels of cry transcription.The geographical distribution of the B1/B2 haplotypes was analyzed, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation.
Yang, S. and Zhang, W. (2022). Systematic analysis of olfactory protein-protein interactions network of fruitfly, Drosophila melanogaster. Arch Insect Biochem Physiol: e21882. PubMed ID: 35249240
Summary:
Olfaction is one of the physiological traits of insect behavior. Insects have evolved a sophisticated olfactory system and use a combined coding strategy to process general odor. Drosophila melanogaster is a powerful model to reveal the molecular and cellular mechanisms of odor detection. Identifying new olfactory targets through complex interactions will contribute to a better understanding of the functions, interactions, and signaling pathways of olfactory proteins. However, the mechanism of D. melanogaster olfaction is still unclear, and more olfactory proteins are required to be discovered. This study tried to explore essential proteins in the olfactory system of D. melanogaster and conduct protein-protein interactions (PPIs) analysis. The PPIs network of the olfactory system of D. melanogaster was constructed, consisting of 863 proteins and 18,959 interactions. Various methods were used to perform functional enrichment analysis, topological analysis and cluster analysis. The results confirmed that Class B scavenger receptors (SR-Bs), glutathione S-transferases (GSTs), and UDP-glycosyltransferases (UGTs) play an essential role in olfaction of D. melanogaster. The proteins obtained in this study can be used for subsequent functional identification in D. melanogaster olfactory system.
Denha, S. A., Atang, A. E., Hays, T. S. and Avery, A. W. (2022). beta-III-spectrin N-terminus is required for high-affinity actin binding and SCA5 neurotoxicity. Sci Rep 12(1): 1726. PubMed ID: 35110634
Summary:
Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). This study investigated in Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). Pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P substitution. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.
Taylor, J. R., Wood, J. G., Mizerak, E., Hinthorn, S., Liu, J., Finn, M., Gordon, S., Zingas, L., Chang, C., Klein, M. A., Denu, J. M., Gorbunova, V., Seluanov, A., Boeke, J. D., Sedivy, J. M. and Helfand, S. L. (2022). Sirt6 regulates lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091469
Summary:
Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. This study characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD(+)-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. These results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.
Zheng, L., Liu, J., Niu, L., Kamran, M., Yang, A. W. H., Jolma, A., Dai, Q., Hughes, T. R., Patel, D. J., Zhang, L., Prasanth, S. G., Yu, Y., Ren, A. and Lai, E. C. (2022). Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins. Genes Dev 36(3-4): 225-240. PubMed ID: 35144965
Summary:
The BEN domain (see Drosophila Insensitive) is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. This study characterized several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. Detailed functional, genomic, and structural studies of BEND3 were conducted. BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. A high-resolution structure was obtained of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Wednesday, June 29th - Larval and Adult Neural Development

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Taylor, J. R., Wood, J. G., Mizerak, E., Hinthorn, S., Liu, J., Finn, M., Gordon, S., Zingas, L., Chang, C., Klein, M. A., Denu, J. M., Gorbunova, V., Seluanov, A., Boeke, J. D., Sedivy, J. M. and Helfand, S. L. (2022). Sirt6 regulates lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091469
Summary:
Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends aging in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. This study we characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD(+)-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. These results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.
Ko, S., Yeom, E., Chun, Y. L., Mun, H., Howard-McGuire, M., Millison, N. T., Jung, J., Lee, K. P., Lee, C., Lee, K. S., Delaney, J. R. and Yoon, J. H. (2022). Profiling of RNA-binding Proteins Interacting With Glucagon and Adipokinetic Hormone mRNAs. J Lipid Atheroscler 11(1): 55-72. PubMed ID: 35118022
Summary:
Glucagon in mammals and its homolog (adipokinetic hormone [AKH] in Drosophila melanogaster) are peptide hormones which regulate lipid metabolism by breaking down triglycerides. Although regulatory mechanisms of glucagon and AKH expression have been widely studied, post-transcriptional gene expression of glucagon has not been investigated thoroughly. This study aimed to profile proteins binding with Gcg messenger RNA (mRNA) in mouse and Akh mRNA in Drosophila. Drosophila Schneider 2 (S2) and mouse 3T3-L1 cell lysates were utilized for affinity pull down of Akh and Gcg mRNA respectively using biotinylated anti-sense DNA oligoes against target mRNAs. Mass spectrometry and computational network analysis revealed mRNA-interacting proteins residing in functional proximity. This study observed that 1) 91 proteins interact with Akh mRNA from S2 cell lysates, 2) 34 proteins interact with Gcg mRNA from 3T3-L1 cell lysates. 3) Akh mRNA interactome revealed clusters of ribosomes and known RNA-binding proteins (RBPs). 4) Gcg mRNA interactome revealed mRNA-binding proteins including Plekha7, zinc finger protein, carboxylase, lipase, histone proteins and a cytochrome, Cyp2c44. 5) Levels of Gcg mRNA and its interacting proteins are elevated in skeletal muscles isolated from old mice compared to ones from young mice.Akh mRNA in S2 cells are under active translation in a complex of RBPs and ribosomes. Gcg mRNA in mouse precursor adipocyte is in a condition distinct from Akh mRNA due to biochemical interactions with a subset of RBPs and histones. It is anticipatet that thiw study contributes to investigating regulatory mechanisms of Gcg and Akh mRNA decay, translation, and localization.
Kamemura, K., Moriya, H., Ukita, Y., Okumura, M., Miura, M. and Chihara, T. (2022). Endoplasmic reticulum proteins Meigo and Gp93 govern dendrite targeting by regulating Toll-6 localization. Dev Biol 484: 30-39. PubMed ID: 35134382
Summary:
Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. This study reports that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, it was found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring.
Srinivasan, A. R., Tran, T. T. and Bonini, N. M. (2022). Loss of miR-34 in Drosophila dysregulates protein translation and protein turnover in the aging brain. Aging Cell 21(3): e13559. PubMed ID: 35166006
Summary:
Aging is a risk factor for neurodegenerative disease, but precise mechanisms that influence this relationship are still under investigation. Work in Drosophila melanogaster identified the microRNA miR-34 as a modifier of aging and neurodegeneration in the brain. MiR-34 mutants present aspects of early aging, including reduced lifespan, neurodegeneration, and a buildup of the repressive histone mark H3K27me3. To better understand how miR-34 regulated pathways contribute to age-associated phenotypes in the brain, here we transcriptionally profiled the miR-34 mutant brain. This identified that genes associated with translation are dysregulated in the miR-34 mutant. The brains of these animals show increased translation activity, accumulation of protein aggregation markers, and altered autophagy activity. To determine if altered H3K27me3 was responsible for this proteostasis dysregulation, the effects of increased H3K27me3 was studied by mutating the histone demethylase Utx. Reduced Utx activity enhanced neurodegeneration and mimicked the protein accumulation seen in miR-34 mutant brains. However, unlike the miR-34 mutant, Utx mutant brains did not show similar altered autophagy or translation activity, suggesting that additional miR-34-targeted pathways are involved. Transcriptional analysis of predicted miR-34 targets identified Lst8, a subunit of Tor Complex 1 (TORC1), as a potential target. We confirmed that miR-34 regulates the 3' UTR of Lst8 and identified several additional predicted miR-34 targets that may be critical for maintaining proteostasis and brain health. Together, these results present novel understanding of the brain and the role of the conserved miRNA miR-34 in impacting proteostasis in the brain with age.
Schell, B., Legrand, P. and Fribourg, S. (2022). Crystal structure of SFPQ-NONO heterodimer. Biochimie 198: 1-7. PubMed ID: 35245601
Summary:
The Drosophila behavior/human splicing (DBHS) protein family is composed of the three members SFPQ, NONO and PSPC1. These proteins share a strong sequence and structural homology within the core-structured domains forming obligate homo- and heterodimers. This feature may lead to the simultaneous existence of six different dimeric complexes that sustain their function in many cellular processes such as pre-mRNA splicing, innate immunity, transcriptional regulation. In order to perform a complete structural analysis of all possible DBHS dimers,this study has solved the crystal structure of the missing DBHS heterodimer SFPQ-NONO at 3.0 Å resolution. Subtle changes were identfied in amino acid composition and local secondary structure of the NOPS region orientation that may modulate affinity between complexes. Interestingly this area is found mutated in aggressive skin cancers and adenocarcinomas.
Yang, N., Srivastav, S. P., Rahman, R., Ma, Q., Dayama, G., Li, S., Chinen, M., Lei, E. P., Rosbash, M. and Lau, N. C. (2022). Transposable element landscapes in aging Drosophila. PLoS Genet 18(3): e1010024. PubMed ID: 35239675
Summary:
Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To address this question, this study quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. TLs were quantified in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when small RNA and RNA interference (RNAi) pathways are compromised. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, it was show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. Our study suggests that in addition to a possible influence by different genetic backgrounds, small RNA and RNAi mechanisms may mitigate genomic TL expansion despite the increase in TE transcripts during aging.

Wednesday, June 30 - Larval and Adult Neural Development and Function

Reinhard, N., Bertolini, E., Saito, A., Sekiguchi, M., Yoshii, T., Rieger, D. and Helfrich-Forster, C. (2022). The lateral posterior clock neurons of Drosophila melanogaster express three neuropeptides and have multiple connections within the circadian clock network and beyond. J Comp Neurol 530(9): 1507-1529. PubMed ID: 34961936
Summary:
Drosophila's lateral posterior neurons (LPNs) belong to a small group of circadian clock neurons that is so far not characterized in detail. Thanks to a new highly specific split-Gal4 line, this study describes LPNs' morphology in fine detail, their synaptic connections, daily bimodal expression of neuropeptides, and a putative role of this cluster is proposed in controlling daily activity and sleep patterns. The three LPNs are heterogeneous. Two of the neurons with similar morphology arborize in the superior medial and lateral protocerebrum and most likely promote sleep. One unique, possibly wakefulness-promoting, neuron with wider arborizations extends from the superior lateral protocerebrum toward the anterior optic tubercle. Both LPN types exhibit manifold connections with the other circadian clock neurons, especially with those that control the flies' morning and evening activity (M- and E-neurons, respectively). In addition, they form synaptic connections with neurons of the mushroom bodies, the fan-shaped body, and with many additional still unidentified neurons. Both LPN types rhythmically express three neuropeptides, Allostatin A, Allostatin C, and Diuretic Hormone 31 with maxima in the morning and the evening. The three LPN neuropeptides may, furthermore, signal to the insect hormonal center in the pars intercerebralis and contribute to rhythmic modulation of metabolism, feeding, and reproduction. These findings are discussed in the light of anatomical details gained by the recently published hemibrain of a single female fly on the electron microscopic level and of previous functional studies concerning the LPN.
Jimeno-Martín, A., Sousa, E., Brocal-Ruiz, R., Daroqui, N., Maicas, M. and Flames, N. (2022). Joint actions of diverse transcription factor families establish neuron-type identities and promote enhancer selectivity.. Genome Res 32(3): 459-473. PubMed ID: 35074859
Summary:
To systematically investigate the complexity of neuron specification regulatory networks, this study carried out an an RNA interference (RNAi) screen against all 875 transcription factors (TFs) encoded in Caenorhabditis elegans genome and searched for defects in nine different neuron types of the monoaminergic (MA) superclass and two cholinergic motoneurons. 91 TF candidates to be required for correct generation of these neuron types, of which 28 were confirmed by mutant analysis. Correct reporter expression in each individual neuron type requires at least nine different TFs. Individual neuron types do not usually share TFs involved in their specification but share a common pattern of TFs belonging to the five most common TF families: homeodomain (HD), basic helix loop helix (bHLH), zinc finger (ZF), basic leucine zipper domain (bZIP), and nuclear hormone receptors (NHR). HD TF members are overrepresented, supporting a key role for this family in the establishment of neuronal identities. These five TF families are also prevalent when considering mutant alleles with previously reported neuronal phenotypes in C. elegans, Drosophila, and mouse. In addition, terminal differentiation complexity was studied focusing on the dopaminergic terminal regulatory program. Two HD TFs (UNC-62 and VAB-3) were found that work together with known dopaminergic terminal selectors (AST-1, CEH-43, CEH-20). Combined TF binding sites for these five TFs constitute a cis-regulatory signature enriched in the regulatory regions of dopaminergic effector genes. These results provide new insights on neuron-type regulatory programs in C. elegans that could help better understand neuron specification and evolution of neuron types.
Lei, Z., Henderson, K. and Keleman, K. (2022). A neural circuit linking learning and sleep in Drosophila long-term memory. Nat Commun 13(1): 609. PubMed ID: 35105888
Summary:
Animals retain some but not all experiences in long-term memory (LTM). Sleep supports LTM retention across animal species. It is well established that learning experiences enhance post-learning sleep. However, the underlying mechanisms of how learning mediates sleep for memory retention are not clear. Drosophila males display increased amounts of sleep after courtship learning. Courtship learning depends on Mushroom Body (MB) neurons, and post-learning sleep is mediated by the sleep-promoting ventral Fan-Shaped Body neurons (vFBs). We show that post-learning sleep is regulated by two opposing output neurons (MBONs) from the MB, which encode a measure of learning. Excitatory MBONs-γ2α'1 becomes increasingly active upon increasing time of learning, whereas inhibitory MBONs-β'2mp is activated only by a short learning experience. These MB outputs are integrated by SFS neurons, which excite vFBs to promote sleep after prolonged but not short training. This circuit may ensure that only longer or more intense learning experiences induce sleep and are thereby consolidated into LTM.
Namiki, S., Ros, I. G., Morrow, C., Rowell, W. J., Card, G. M., Korff, W. and Dickinson, M. H. (2022). A population of descending neurons that regulates the flight motor of Drosophila. Curr Biol 32(5): 1189-1196. PubMed ID: 35090590
Summary:
Similar to many insect species, Drosophila melanogaster is capable of maintaining a stable flight trajectory for periods lasting up to several hours. Because aerodynamic torque is roughly proportional to the fifth power of wing length, even small asymmetries in wing size require the maintenance of subtle bilateral differences in flapping motion to maintain a stable path. Flies can even fly straight after losing half of a wing, a feat they accomplish via very large, sustained kinematic changes to both the damaged and intact wings. This study describes an unusual type of descending neuron (DNg02) that projects directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike many descending neurons, which exist as single bilateral pairs with unique morphology, there is a population of at least 15 DNg02 cell pairs with nearly identical shape. By optogenetically activating different numbers of DNg02 cells, this study demonstrated that these neurons regulate wingbeat amplitude over a wide dynamic range via a population code. Using two-photon functional imaging, it was shown that DNg02 cells are responsive to visual motion during flight in a manner that would make them well suited to continuously regulate bilateral changes in wing kinematics.
Sato, K. and Yamamoto, D. (2022). Mutually exclusive expression of sex-specific and non-sex-specific fruitless gene products in the Drosophila central nervous system. Gene Expr Patterns 43: 119232. PubMed ID: 35124238
Summary:
The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, this study compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. Nearly all neuroblasts were found to express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons.
Zhu, H., Zhao, S. D., Ray, A., Zhang, Y. and Li, X. (2022). A comprehensive temporal patterning gene network in Drosophila medulla neuroblasts revealed by single-cell RNA sequencing. Nat Commun 13(1): 1247. PubMed ID: 35273186
Summary:
During development, neural progenitors are temporally patterned to sequentially generate a variety of neural types. In Drosophila neural progenitors called neuroblasts, temporal patterning is regulated by cascades of Temporal Transcription Factors (TTFs). However, known TTFs were mostly identified through candidate approaches and may not be complete. In addition, many fundamental questions remain concerning the TTF cascade initiation, progression, and termination. This work used single-cell RNA sequencing of Drosophila medulla neuroblasts of all ages to identify a list of previously unknown TTFs, and experimentally characterize their roles in temporal patterning and neuronal specification. This study reveals a comprehensive temporal gene network that patterns medulla neuroblasts from start to end. Furthermore, the speed of the cascade progression is regulated by Lola transcription factors expressed in all medulla neuroblasts. Our comprehensive study of the medulla neuroblast temporal cascade illustrates mechanisms that may be conserved in the temporal patterning of neural progenitors.

Tuesday, June 28th - Adult Development

Nikonova, E., Mukherjee, A., Kamble, K., Barz, C., Nongthomba, U. and Spletter, M. L. (2022). Rbfox1 is required for myofibril development and maintaining fiber type-specific isoform expression in Drosophila muscles. Life Sci Alliance 5(4). PubMed ID: 34996845
Summary:
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. This study reports that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
Jauregui-Lozano, J., Hall, H., Stanhope, S. C., Bakhle, K., Marlin, M. M. and Weake, V. M. (2022). The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress. PLoS Genet 18(1): e1010021. PubMed ID: 35100266
Summary:
The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. This study integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When the Clock:Cycle complex was disrupted by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, changes were observed in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, expression of ClkDN in photoreceptors was shown to lead to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, these data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress.
Morin-Poulard, I., Destalminil-Letourneau, M., Bataille, L., Frendo, J. L., Lebreton, G., Vanzo, N. and Crozatier, M. (2022). Identification of Bipotential Blood Cell/Nephrocyte Progenitors in Drosophila: Another Route for Generating Blood Progenitors. Front Cell Dev Biol 10: 834720. PubMed ID: 35237606
Summary:
The Drosophila lymph gland is the larval hematopoietic organ and is aligned along the anterior part of the cardiovascular system, composed of cardiac cells, that form the cardiac tube and its associated pericardial cells or nephrocytes. By the end of embryogenesis the lymph gland is composed of a single pair of lobes. Two additional pairs of posterior lobes develop during larval development to contribute to the mature lymph gland. This study describes the ontogeny of lymph gland posterior lobes during larval development and identifies the genetic basis of the process. By lineage tracing it was shown that each posterior lobe originates from three embryonic pericardial cells, thus establishing a bivalent blood cell/nephrocyte potential for a subset of embryonic pericardial cells. The posterior lobes of L3 larvae posterior lobes are composed of heterogeneous blood progenitors and their diversity is progressively built during larval development. It was further established that in larvae, homeotic genes and the transcription factor Klf15 regulate the choice between blood cell and nephrocyte fates. These data underline the sequential production of blood cell progenitors during larval development.
Dean, D. M., Deitcher, D. L., Paster, C. O., Xu, M. and Loehlin, D. W. (2022). "A fly appeared": sable, a classic Drosophila mutation, maps to Yippee, a gene affecting body color, wings, and bristles. G3 (Bethesda). PubMed ID: 35266526
Summary:
Insect body color is an easily assessed and visually engaging trait that is informative on a broad range of topics including speciation, biomaterial science, and ecdysis. Mutants of the fruit fly Drosophila melanogaster have been an integral part of body color research for more than a century. As a result of this long tenure, backlogs of body color mutations have remained unmapped to their genes, all while their strains have been dutifully maintained, used for recombination mapping, and part of genetics education. Stemming from a lesson plan in our undergraduate genetics class, we have mapped sable1, a dark body mutation originally described by Morgan and Bridges, to Yippee, a gene encoding a predicted member of the E3 ubiquitin ligase complex. Deficiency/duplication mapping, genetic rescue, DNA and cDNA sequencing, RT-qPCR, and two new CRISPR alleles indicated that sable1 is a hypomorphic Yippee mutation due to a mdg4 element insertion in the Yippee 5'-UTR. Further analysis revealed additional Yippee mutant phenotypes including curved wings, ectopic/missing bristles, delayed development, and failed adult emergence. RNAi of Yippee in the ectoderm phenocopied sable body color and most other Yippee phenotypes. Although Yippee remains functionally uncharacterized, the results presented this study suggest possible connections between melanin biosynthesis, copper homeostasis, and Notch/Delta signaling; in addition, they provide insight into past studies of sable cell nonautonomy and of the genetic modifier suppressor of sable.
Fang, C., Xin, Y., Sun, T., Monteiro, A., Ye, Z., Dai, F., Lu, C. and Tong, X. (2022). The Hox gene Antennapedia is essential for wing development in insects. Development 149(2). PubMed ID: 35088829
Summary:
A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. This study shows that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. It is proposed, therefore, that insect wing development occurs in an Antp-dependent manner.
Wang, H., Morrison, C. A., Ghosh, N., Tea, J. S., Call, G. B. and Treisman, J. E. (2022). The Blimp-1 transcription factor acts in non-neuronal cells to regulate terminal differentiation of the Drosophila eye. Development 149(7). PubMed ID: 35297965
Summary:
The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. This study shows that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.

Monday June 27th - Evolution

Loehlin, D. W., Kim, J. Y. and Paster, C. O. (2022). A tandem duplication in Drosophila melanogaster shows enhanced expression beyond the gene copy number. Genetics 220(3). PubMed ID: 35100388
Summary:
Tandem duplicated genes are common features of genomes, but the phenotypic consequences of their origins are not well understood. It is not known whether a simple doubling of gene expression should be expected, or else some other expression outcome. This study describes an experimental framework using engineered deletions to assess any contribution of locally acting cis- and globally acting trans-regulatory factors to expression interactions of particular tandem duplicated genes. Acsx1L (CG6300) and Acsx1R (CG11659) are tandem duplicates of a putative acyl-CoA synthetase gene found in Drosophila melanogaster. Experimental deletions of the duplicated segments were used to investigate whether the presence of 1 tandem duplicated block influences the expression of its neighbor. Acsx1L, the gene in the left block, shows much higher expression than either its duplicate Acsx1R or the single Acsx1 in Drosophila simulans. Acsx1L expression decreases drastically upon deleting the right-hand duplicated block. Crosses among wildtype and deletion strains show that high tandem expression is primarily due to cis-acting interactions between the duplicated blocks. No effect of these genes on cuticular hydrocarbons was detected. Sequence and phylogenetic analysis suggest that the duplication rose to fixation in D. melanogaster and has been subject to extensive gene conversion. Some strains actually carry 3 tandem copies, yet strains with 3 Acsx1s do not have higher expression levels than strains with 2. Surveys of tandem duplicate expression have typically not found the expected 2-fold increase in expression. This study suggests that cis-regulatory interactions between duplicated blocks could be responsible for this trend.
Hakeemi, M. S., Ansari, S., Teuscher, M., Weisskopf, M., Grossmann, D., Kessel, T., Dönitz, J., Siemanowski, J., Wan, X., Schultheis, D., Frasch, M., Roth, S., Schoppmeier, M., Klingler, M. and Bucher, G. (2022). Screens in fly and beetle reveal vastly divergent gene sets required for developmental processes. BMC Biol 20(1): 38. PubMed ID: 35135533
Summary:
Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative-to some degree-for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. This study systematically compared the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded an RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (~ 10%) or beetle (~ 40%) reflecting both technical and biological differences. Accordingly, it was possible to annotate novel developmental GO terms for 96 genes studied in this work. This work is the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). It is concluded that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor.
Boitard, S., Arredondo, A., Chikhi, L. and Mazet, O. (2022). Heterogeneity in effective size across the genome: effects on the inverse instantaneous coalescence rate (IICR) and implications for demographic inference under linked selection. Genetics 220(3). PubMed ID: 35100421
Summary:
The relative contribution of selection and neutrality in shaping species genetic diversity is one of the most central and controversial questions in evolutionary theory. Genomic data provide growing evidence that linked selection, i.e. the modification of genetic diversity at neutral sites through linkage with selected sites, might be pervasive over the genome. Several studies proposed that linked selection could be modeled as first approximation by a local reduction (e.g. purifying selection, selective sweeps) or increase (e.g. balancing selection) of effective population size (Ne). At the genome-wide scale, this leads to variations of Ne from one region to another, reflecting the heterogeneity of selective constraints and recombination rates between regions. This study investigated the consequences of such genomic variations of Ne on the genome-wide distribution of coalescence times. The underlying motivation concerns the impact of linked selection on demographic inference, because the distribution of coalescence times is at the heart of several important demographic inference approaches. Using the concept of inverse instantaneous coalescence rate, it was demonstrated that in a panmictic population, linked selection always results in a spurious apparent decrease of Ne along time. Balancing selection has a particularly large effect, even when it concerns a very small part of the genome. More general models were studied including genuine population size changes, population structure or transient selection, and the effect of linked selection was found to be significantly reduced by that of population structure. The models and conclusions presented in this study are also relevant to the study of other biological processes generating apparent variations of Ne along the genome.
Lange, J. D., Bastide, H., Lack, J. B. and Pool, J. E. (2022). A Population Genomic Assessment of Three Decades of Evolution in a Natural Drosophila Population. Mol Biol Evol 39(2). PubMed ID: 34971382
Summary:
Population genetics seeks to illuminate the forces shaping genetic variation, often based on a single snapshot of genomic variation. However, utilizing multiple sampling times to study changes in allele frequencies can help clarify the relative roles of neutral and non-neutral forces on short time scales. This study compares whole-genome sequence variation of recently collected natural population samples of Drosophila melanogaster against a collection made approximately 35 years prior from the same locality-encompassing roughly 500 generations of evolution. The allele frequency changes between these time points would suggest a relatively small local effective population size on the order of 10,000, significantly smaller than the global effective population size of the species. Some loci display stronger allele frequency changes than would be expected anywhere in the genome under neutrality-most notably the tandem paralogs Cyp6a17 and Cyp6a23, which are impacted by structural variation associated with resistance to pyrethroid insecticides. A genome-wide excess of outliers was found for high genetic differentiation between old and new samples, but a larger number of adaptation targets may have affected SNP-level differentiation versus window differentiation. Evidence was found for strengthening latitudinal allele frequency clines: northern-associated alleles have increased in frequency by an average of nearly 2.5% at SNPs previously identified as clinal outliers, but no such pattern is observed at random SNPs. This project underscores the scientific potential of using multiple sampling time points to investigate how evolution operates in natural populations, by quantifying how genetic variation has changed over ecologically relevant timescales.
Matsunaga, T., Reisenman, C. E., Goldman-Huertas, B., Brand, P., Miao, K., Suzuki, H. C., Verster, K. I., Ramirez, S. R. and Whiteman, N. K. (2022). Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae. Mol Biol Evol 39(2). PubMed ID: 34963012
Summary:
The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. This study focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, this study found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.
Janssen, R. and Budd, G. E. (2022). Expression of netrin and its receptors uncoordinated-5 and frazzled in arthropods and onychophorans suggests conserved and diverged functions in neuronal pathfinding and synaptogenesis. Dev Dyn. PubMed ID: 35112412
Summary:
Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane-bound receptors such as Uncoordinated-5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. This study investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated-5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. The expression patterns were also studied in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and data is provided on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. These data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages.

Friday, June 24th - Adult Physiology

Walsh, B. S., Parratt, S. R., Mannion, N. L. M., Snook, R. R., Bretman, A. and Price, T. A. R. (2021). Plastic responses of survival and fertility following heat stress in pupal and adult Drosophila virilis. Ecol Evol 11(24): 18238-18247. PubMed ID: 35003670
Summary:
The impact of rising global temperatures on survival and reproduction is putting many species at risk of extinction. In particular, it has recently been shown that thermal effects on reproduction, especially limits to male fertility, can underpin species distributions in insects. However, the physiological factors influencing fertility at high temperatures are poorly understood. Key factors that affect somatic thermal tolerance such as hardening, the ability to phenotypically increase thermal tolerance after a mild heat shock, and the differential impact of temperature on different life stages are largely unexplored for thermal fertility tolerance. This study examine the impact of high temperatures on male fertility in the cosmopolitan fruit fly Drosophila virilis. It was first determined whether temperature stress at either the pupal or adult life history stage impacts fertility. Then the capacity for heat-hardening to mitigate heat-induced sterility was tested. Thermal stress was found to reduce fertility in different ways in pupae and adults. Pupal heat stress delays sexual maturity, whereas males heated as adults can reproduce initially following heat stress, but become sterile within seven days. Evidence was also found that while heat-hardening in D. virilis can improve high temperature survival, there is no significant protective impact of this same hardening treatment on fertility. These results suggest that males may be unable to prevent the costs of high temperature stress on fertility through heat-hardening, which limits a species' ability to quickly and effectively reduce fertility loss in the face of short-term high temperature events.
Bai, Y. and Suzuki, T. (2022). Activity-dependent circuitry plasticity via the regulation of the histamine receptor level in the Drosophila visual system. Mol Cell Neurosci 119: 103703. PubMed ID: 35122941
Summary:
Activity-dependent synaptic plasticity is crucial for responses to the environment. Although the plasticity mechanisms of presynaptic photoreceptor neurons in the Drosophila visual system have been well studied, postsynaptic modifications remain elusive. In addition, further studies on the adaption of the visual system to different light experiences at a circuitry scale are required. Using the modified transcriptional reporter of intracellular Ca(2+) method, this study describes a way to visualize circuitry changes according to different light experiences. Enhanced postsynaptic neuronal activity responses were found in lamina monopolar neuron L2 after prolonged light treatment. Although L1 also has connections with photoreceptors, there were no enhanced activity responses in L1. Activity-dependent transcriptional downregulation of inhibitory histamine receptor (HR), Ort, was found to occur in postsynaptic neuron L2, but not in L1, during continuous light conditions. Exogenous Ort proteins were produced in L2 neurons and they were found to attenuate the enhanced activity response caused by constant light exposure. These findings, together with the fact that histamine is the main inhibitory neurotransmitter released by photoreceptors in the Drosophila visual system, confirmed the hypothesis that the activity-dependent transcriptional downregulation of HR is responsible for the constant light exposure-induced circuitry response changes in L2. The results successfully demonstrated the selective circuit change after synaptic remodeling evoked by long-term activation and provided in vivo evidence of circuitry plasticity upon long-term environmental stimulation.
Ueda, K., Anderson-Baron, M. N., Haskins, J., Hughes, S. C. and Simmonds, A. J. (2022). Recruitment of Peroxin 14 to lipid droplets affects lipid storage in Drosophila. J Cell Sci 135(7). PubMed ID: 35274690
Summary:
Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. This study has shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage.
Jones, E. W., Carlson, J. M., Sivak, D. A. and Ludington, W. B. (2022). Stochastic microbiome assembly depends on context. Proc Natl Acad Sci U S A 119(7). PubMed ID: 35135881
Summary:
Observational studies reveal substantial variability in microbiome composition across individuals. Targeted studies in gnotobiotic animals underscore this variability by showing that some bacterial strains colonize deterministically, while others colonize stochastically. While some of this variability can be explained by external factors like environmental, dietary, and genetic differences between individuals, this paper shows that for the model organism Drosophila melanogaster, interactions between bacteria can affect the microbiome assembly process, contributing to a baseline level of microbiome variability even among isogenic organisms that are identically reared, housed, and fed. In germ-free flies fed known combinations of bacterial species, some species were found to colonize more frequently than others even when fed at the same high concentration. This study developed an ecological technique that infers the presence of interactions between bacterial species based on their colonization odds in different contexts, requiring only presence/absence data from two-species experiments. A progressive sequence of probabilistic models, in which the colonization of each bacterial species is treated as an independent stochastic process, was used to reproduce the empirical distributions of colonization outcomes across experiments. Incorporating context-dependent interactions substantially was found to improve the performance of the models. Stochastic, context-dependent microbiome assembly underlies clinical therapies like fecal microbiota transplantation and probiotic administration and should inform the design of synthetic fecal transplants and dosing regimes.
Krittika, S. and Yadav, P. (2022). Trans-generational effect of protein restricted diet on adult body and wing size of Drosophila melanogaster. R Soc Open Sci 9(1): 211325. PubMed ID: 35116152
Summary:
Protein restriction (PR) has established feasible trade-offs in Drosophila melanogaster to understand lifespan or ageing in a nutritionally challenged environment. However, the phenotypes of body size, weight and wing length respond according to factors such as flies' genotype, environmental exposure and parental diet, and hence their understanding is essential. This study demonstrates the effect of long-term PR diet on body size, weight, normal and dry wing length of flies subjected to PR50 and PR70 (50% and 70% protein content present in control food, respectively) for 20 generations from the pre-adult stage. PR-fed flies were found to have lower body weight, relative water content (in males), unaltered (PR50%) and higher (PR70%) relative fat content in males, smaller normal and dry body size when compared with control and generations 1 and 2. Interestingly, the wing size and pupal size of PR flies are smaller and showed significant effects on diet and generation. Thus, these traits are sex and generation dependent along with a diet interaction, which is capable of modulating these results variably. Taken together, the trans-generational effect of PR on fitness and fitness-related traits might be helpful to understand the underpinning mechanisms of evolution and ageing in fruit flies D. melanogaster.
Yang, P., Yang, X., Sun, L., Han, X., Xu, L., Gu, W. and Zhang, M. (2022). Effects of cadmium on oxidative stress and cell apoptosis in Drosophila melanogaster larvae. Sci Rep 12(1): 4762. PubMed ID: 35307728
Summary:
With the increase of human activities, cadmium (Cd) pollution has become a global environmental problem affecting biological metabolism in ecosystem. Cd has a very long half-life in humans and is excreted slowly in organs, which poses a serious threat to human health. In order to better understand the toxicity effects of cadmium, third instar larvae of Drosophila melanogaster (Canton-S strain) were exposed to different concentrations (1.125 mg/kg, 2.25 mg/kg, 4.5 mg/kg, and 9 mg/kg) of cadmium. Trypan blue staining showed that intestinal cell damage of Drosophila larvae increased and the comet assay indicated significantly more DNA damage in larvae exposed to high Cd concentrations. The nitroblue tetrazolium (NBT) experiments proved that content of reactive oxygen species (ROS) increased, which indicated Cd exposure could induce oxidative stress. In addition, the expression of mitochondrial adenine nucleotide transferase coding gene (sesB and Ant2) and apoptosis related genes (Debcl, hid, rpr, p53, Sce and Diap1) changed, which may lead to increased apoptosis. These findings confirmed the toxicity effects on oxidative stress and cell apoptosis in Drosophila larvae after early cadmium exposure, providing insights into understanding the effects of heavy metal stress in animal development.

Thursday, June 23rd - Adult neural development and function

Stahl, A., Noyes, N. C., Boto, T., Botero, V., Broyles, C. N., Jing, M., Zeng, J., King, L. B., Li, Y., Davis, R. L. and Tomchik, S. M. (2022). Associative learning drives longitudinally graded presynaptic plasticity of neurotransmitter release along axonal compartments. Elife 11. PubMed ID: 35285796
Summary:
Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. This study shows that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the Ca(V)2.1 calcium channel subunit Cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor, which maintained responsivity to odors upon repeated presentations, preventing adaptation. Downstream from the MB, a set of MBONs that receive their input from the γ3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which reward learning influences decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation, and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit.
Hakeemi, M. S., Ansari, S., Teuscher, M., Weisskopf, M., Grossmann, D., Kessel, T., Dönitz, J., Siemanowski, J., Wan, X., Schultheis, D., Frasch, M., Roth, S., Schoppmeier, M., Klingler, M. and Bucher, G. (2022). Screens in fly and beetle reveal vastly divergent gene sets required for developmental processes. BMC Biol 20(1): 38. PubMed ID: 35135533
Summary:
Drosophila is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. This study systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, the RNAi screen in the red flour beetle Tribolium castaneum was expandec to cover more than half of the protein-coding genes. The gene sets required for four different developmental processes between beetle and fly were compared. Around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (~ 10%) or beetle (~ 40%) reflecting both technical and biological differences. Accordingly, it was possible to annotate novel developmental GO terms for 96 genes studied in this work. This paper is the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). It is concluded that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor.
Trisal, S., Aranha, M., Chodankar, A., VijayRaghavan, K. and Ramaswami, M. (2022). A Drosophila Circuit for Habituation Override. J Neurosci 42(14): 2930-2941. PubMed ID: 35232763
Summary:
Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila. This study analyzed how exposure to a novel stimulus causes override of gustatory (proboscis extension reflex; PER) habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. This study shows that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of tyrosine hydroxylase (TH)-positive neurons, a subset of which send projections to the subesophageal zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.
Bai, Y. and Suzuki, T. (2022). Activity-dependent circuitry plasticity via the regulation of the histamine receptor level in the Drosophila visual system. Mol Cell Neurosci 119: 103703. PubMed ID: 35122941
Summary:
Activity-dependent synaptic plasticity is crucial for responses to the environment. Although the plasticity mechanisms of presynaptic photoreceptor neurons in the Drosophila visual system have been well studied, postsynaptic modifications remain elusive. In addition, further studies on the adaption of the visual system to different light experiences at a circuitry scale are required. Using the modified transcriptional reporter of intracellular Ca(2+) method, this study describes a way to visualize circuitry changes according to different light experiences. Enhanced postsynaptic neuronal activity responses were found in lamina monopolar neuron L2 after prolonged light treatment. Although L1 also has connections with photoreceptors, there were no enhanced activity responses in L1. This study also reports that activity-dependent transcriptional downregulation of inhibitory histamine receptor (HR), Ort, occurs in postsynaptic neuron L2, but not in L1, during continuous light conditions. This study produced exogenous Ort proteins in L2 neurons and found that it attenuated the enhanced activity response caused by constant light exposure. These findings, together with the fact that histamine is the main inhibitory neurotransmitter released by photoreceptors in the Drosophila visual system, confirmed our hypothesis that the activity-dependent transcriptional downregulation of HR is responsible for the constant light exposure-induced circuitry response changes in L2. The results successfully demonstrated the selective circuit change after synaptic remodeling evoked by long-term activation and provided in vivo evidence of circuitry plasticity upon long-term environmental stimulation.
Chen, R., Deng, X. and Zhu, S. (2022). The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless. PLoS Genet 18(1): e1009928. PubMed ID: 35100262
Summary:
Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. This study utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. The results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. This study further showa that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, thus this study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1.
Israel, S., Rozenfeld, E., Weber, D., Huetteroth, W. and Parnas, M. (2022). Olfactory stimuli and moonwalker SEZ neurons can drive backward locomotion in Drosophila. Curr Biol 32(5): 1131-1149. PubMed ID: 35139358
Summary:
How different sensory stimuli are collected, processed, and further transformed into a coordinated motor response is a fundamental question in neuroscience. In particular, the internal and external conditions that drive animals to switch to backward walking and the mechanisms by which the nervous system supports such behavior are still unknown. In fruit flies, moonwalker descending neurons (MDNs) are considered command-type neurons for backward locomotion as they receive visual and mechanosensory inputs and transmit motor-related signals to downstream neurons to elicit backward locomotion. Whether other modalities converge onto MDNs, which central brain neurons activate MDNs, and whether other retreat-driving pathways exist is currently unknown. Here, we show that olfactory stimulation can elicit MDN-mediated backward locomotion. Moreover, this study identified the moonwalker subesophageal zone neurons (MooSEZs), a pair of bilateral neurons, which can trigger straight and rotational backward locomotion. MooSEZs act via postsynaptic MDNs and via other descending neurons. Although they respond to olfactory input, they are not required for odor-induced backward walking. Thus, this work reveals an important modality input to MDNs, a novel set of neurons presynaptic to MDNs driving backward locomotion and an MDN-independent backward locomotion pathway.

Wednesday June 22nd - Disease Models

Jauregui-Lozano, J., Escobedo, S., Easton, A., Lanman, N. A., Weake, V. M. and Hall, H. (2022). Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging. Aging Cell 21(2): e13554. PubMed ID: 35048512
Summary:
Age-related loss of cellular function and increased cell death are characteristic hallmarks of aging. While defects in gene expression and RNA metabolism have been linked with age-associated human neuropathies, it is not clear how the changes that occur in aging neurons contribute to loss of gene expression homeostasis. R-loops are RNA-DNA hybrids that typically form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been associated with both transcriptional impairment and genome instability. Importantly, a growing body of evidence links R-loop accumulation with cellular dysfunction, increased cell death, and chronic disease onset. This study characterized the R-loop landscape in aging Drosophila melanogaster photoreceptor neurons and showed that bulk R-loop levels increased with age. Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R-loops over gene bodies during aging, which correlated with decreased expression of long and highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, over-expression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging. Together, these studies highlight the functional link between dysregulation of R-loop homeostasis, gene expression, and visual function during aging.
Gerstner, L., Chen, M., Kampf, L. L., Milosavljevic, J., Lang, K., Schneider, R., Hildebrandt, F., Helmstadter, M., Walz, G. and Hermle, T. (2022). Inhibition of endoplasmic reticulum stress signaling rescues cytotoxicity of human apolipoprotein-L1 risk variants in Drosophila. Kidney Int. PubMed ID: 35120995
Summary:
Risk variants of the apolipoprotein-L1 (APOL1) gene are associated with severe kidney disease, putting homozygous carriers at risk. Since APOL1 lacks orthologs in all major model organisms, a wide range of mechanisms frequently in conflict have been described for APOL1-associated nephropathies. The genetic toolkit in Drosophila allows unique in vivo insights into disrupted cellular homeostasis. To perform a mechanistic analysis, this study expressed human APOL1 control and gain-of-function kidney risk variants in the podocyte-like garland cells of Drosophila nephrocytes and a wing precursor tissue. Expression of APOL1 risk variants was found to elevate endocytic function of garland cell nephrocytes that simultaneously showed early signs of cell death. Wild-type APOL1 had a significantly milder effect, while a control transgene with deletion of the short BH3 domain showed no overt phenotype. Nephrocyte endo-lysosomal function and slit diaphragm architecture remained unaffected by APOL1 risk variants, but endoplasmic reticulum (ER) swelling, chaperone induction, and expression of the reporter Xbp1-EGFP suggested an ER stress response. Pharmacological inhibition of ER stress diminished APOL1-mediated cell death and direct ER stress induction enhanced nephrocyte endocytic function similar to expression of APOL1 risk variants. APOL1-dependent ER stress was cofirmed in the Drosophila wing precursor where silencing the IRE1-dependent branch of ER stress signaling by inhibition with Xbp1-RNAi abrogated cell death, representing the first rescue of APOL1-associated cytotoxicity in vivo. Thus, this study uncovered ER stress as an essential consequence of APOL1 risk variant expression in vivo in Drosophila, suggesting a central role of this pathway in the pathogenesis of APOL1-associated nephropathies.
Deolankar, S. C., Najar, M. A., Raghu, S. V. and Prasad, T. S. K. (2022). Abeta42 Expressing Drosophila melanogaster Model for Alzheimer's Disease: Quantitative Proteomics Identifies Altered Protein Dynamics of Relevance to Neurodegeneration. Omics 26(1): 51-63. PubMed ID: 35006003
Summary:
Production and deposition of β-amyloid peptides (Aβ) are among the major hallmarks of the pathogenesis of Alzheimer's disease (AD). Mapping the altered protein dynamics associated with Aβ accumulation and neuronal damage may open up new avenues to innovation for drug target discovery in AD. Using quantitative proteomics, new findings are reported from the amyloid beta-peptide with 42 amino acids (Aβ42) expressing Drosophila melanogaster model for AD compared to that of the wild-type flies. 302,241 peptide-spectrum matches were identified with 25,641 nonredundant peptides corresponding to 7959 D. melanogaster proteins. Furthermore, 538 significantly altered proteins were unraveled in Aβ42 expressing flies. These differentially expressed proteins were enriched for biological processes associated with neuronal damage leading to AD progression. 463 unique post-translational modification events mapping to 202 proteins were identified from the same dataset. Among these, 303 modified peptides corresponding to 246 proteins were also altered in the AD model. These modified proteins are known to be involved in the disruption of molecular functions maintaining neuronal plasticity. This study provides new molecular leads on altered protein dynamics relevant to neurodegeneration, neuroplasticity, and AD progression induced by Aβ42 toxicity. These proteins may prove useful to discover new drugs in an AD model of D. melanogaster and evaluate their efficacy and mode of molecular action in the future.
Wu, Q., Akhter, A., Pant, S., Cho, E., Zhu, J. X., Garner, A., Ohyama, T., Tajkhorshid, E., van Meyel, D. J. and Ryan, R. M. (2022). Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function. J Clin Invest 132(7). PubMed ID: 35167492
Summary:
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. This study investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes and on CNS function in a Drosophila melanogaster model of locomotor behavior. These results indicate that mutations resulting in decreased hEAAT1 Cl- channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. This study also identified what is believed to be a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.
Yang, N., Srivastav, S. P., Rahman, R., Ma, Q., Dayama, G., Li, S., Chinen, M., Lei, E. P., Rosbash, M. and Lau, N. C. (2022). Transposable element landscapes in aging Drosophila. PLoS Genet 18(3): e1010024. PubMed ID: 35239675
Summary:
Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To address this question, TE Landscapes (TLs) were quantified via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. TLs were quantified in whole flies and dissected brains, and the feasibility of this approach was validated in detecting new TE insertions in aging Drosophila genomes when small RNA and RNA interference (RNAi) pathways are compromised. This study also describes improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, it was shown that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. This study suggests that in addition to a possible influence by different genetic backgrounds, small RNA and RNAi mechanisms may mitigate genomic TL expansion despite the increase in TE transcripts during aging.
Zhang, Y., Lin, S., Peng, J., Liang, X., Yang, Q., Bai, X., Li, Y., Li, J., Dong, W., Wang, Y., Huang, Y., Pei, Y., Guo, J., Zhao, W., Zhang, Z., Liu, M. and Zhu, A. J. (2022). Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination. Mol Cell. PubMed ID: 35245436
Summary:
Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. In this study, using Drosophila oenocytes, the specialized hepatocyte-like cells, dietary essential amino acids were found to ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. This study further showed that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, these results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

Tuesday, June 21st - Synapse and Vesicles

Nath, A. S., Parsons, B. D., Makdissi, S., Chilvers, R. L., Mu, Y., Weaver, C. M., Euodia, I., Fitze, K. A., Long, J., Scur, M., Mackenzie, D. P., Makrigiannis, A. P., Pichaud, N., Boudreau, L. H., Simmonds, A. J., Webber, C. A., Derfalvi, B., Hammon, Y., Rachubinski, R. A. and Di Cara, F. (2022). Modulation of the cell membrane lipid milieu by peroxisomal beta-oxidation induces Rho1 signaling to trigger inflammatory responses. Cell Rep 38(9): 110433. PubMed ID: 35235794
Summary:
Phagocytosis, signal transduction, and inflammatory responses require changes in lipid metabolism. Peroxisome have key roles in fatty acid homeostasis and in regulating immune function. Drosophila macrophages lacking peroxisomes have perturbed lipid profiles, which reduce host survival after infection. Using lipidomic, transcriptomic, and genetic screens, we determine that peroxisomes contribute to the cell membrane glycerophospholipid composition necessary to induce Rho1-dependent signals, which drive cytoskeletal remodeling during macrophage activation. Loss of peroxisome function increases membrane phosphatidic acid (PA) and recruits RhoGAPp190 during infection, inhibiting Rho1-mediated responses. Peroxisome-glycerophospholipid-Rho1 signaling also controls cytoskeleton remodeling in mouse immune cells. While high levels of PA in cells without peroxisomes inhibit inflammatory phenotypes, large numbers of peroxisomes and low amounts of cell membrane PA are features of immune cells from patients with inflammatory Kawasaki disease and juvenile idiopathic arthritis. These findings reveal potential metabolic markers and therapeutic targets for immune diseases and metabolic disorders.
Cho, T. S., Beigaite, E., Klein, N. E., Sweeney, S. T. and Bhattacharya, M. R. C. (2022). The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction. Mol Neurobiol. PubMed ID: 35107803
Summary:
TMEM184B is a putative seven-pass membrane protein that promotes axon degeneration after injury. TMEM184B mutation causes aberrant neuromuscular architecture and sensory and motor behavioral defects in mice. The mechanism through which TMEM184B causes neuromuscular defects is unknown. We employed Drosophila melanogaster to investigate the function of the closely related gene, Tmep (CG12004), at the neuromuscular junction. Tmep was shown to be required for full adult viability and efficient larval locomotion. Tmep mutant larvae have a reduced body contraction rate compared to controls, with stronger deficits in females. In recordings from body wall muscles, Tmep mutants show substantial hyperexcitability, with many postsynaptic potentials fired in response to a single stimulation, consistent with a role for Tmep in restraining synaptic excitability. Additional branches and satellite boutons at Tmep mutant neuromuscular junctions are consistent with an activity-dependent synaptic overgrowth. Tmep is expressed in endosomes and synaptic vesicles within motor neurons, suggesting a possible role in synaptic membrane trafficking. Using RNAi knockdown, this study show that Tmep is required in motor neurons for proper larval locomotion and excitability, and that its reduction increases levels of presynaptic calcium. Locomotor defects can be rescued by presynaptic knockdown of endoplasmic reticulum calcium channels or by reducing evoked release probability, further suggesting that excess synaptic activity drives behavioral deficiencies. This work establishes a critical function for Tmep in the regulation of synaptic transmission and locomotor behavior.
Nassari, S., Lacarriere-Keita, C., Levesque, D., Boisvert, F. M. and Jean, S. (2022). Rab21 in enterocytes participates in intestinal epithelium maintenance. Mol Biol Cell 33(4): ar32. PubMed ID: 35171715
Summary:
Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery.This study focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, this study identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. It was further shown that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, changes were validated in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. These data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance.
Certel, S. J., Ruchti, E., McCabe, B. D. and Stowers, R. S. (2022). A conditional glutamatergic synaptic vesicle marker for Drosophila. G3 (Bethesda) 12(3). PubMed ID: 35100385
Summary:
Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development, as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. This study described and characterized smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut was validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver.
Schnute, B., Shimizu, H., Lyga, M., Baron, M. and Klein, T. (2022). Ubiquitylation is required for the incorporation of the Notch receptor into intraluminal vesicles to prevent prolonged and ligand-independent activation of the pathway. BMC Biol 20(1): 65. PubMed ID: 35264151
Summary:
Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. This study analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. A analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. Two functions were identifiee for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. These findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms.
Lambert, E., Saha, O., Soares Landeira, B., Melo de Farias, A. R., Hermant, X., Carrier, A., Pelletier, A., Gadaut, J., Davoine, L., Dupont, C., Amouyel, P., Bonnefond, A., Lafont, F., Abdelfettah, F., Verstreken, P., Chapuis, J., Barois, N., Delahaye, F., Dermaut, B., Lambert, J. C., Costa, M. R. and Dourlen, P. (2022). The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects. Acta Neuropathol Commun 10(1): 4. PubMed ID: 34998435
Summary:
The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. This study observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, thee data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

Friday, June 17th - Enhancers and Transcriptional Regulation

Luecke, D., Rice, G. and Kopp, A. (2022). Sex-specific evolution of a Drosophila sensory system via interacting cis- and trans-regulatory changes. Evol Dev 24(1-2): 37-60. PubMed ID: 35239254
Summary:
The evolution of gene expression via cis-regulatory changes is well established as a major driver of phenotypic evolution. However, relatively little is known about the influence of enhancer architecture and intergenic interactions on regulatory evolution. This question was addressed by examining chemosensory system evolution in Drosophila. Drosophila prolongata males show a massively increased number of chemosensory bristles compared to females and males of sibling species. This increase is driven by sex-specific transformation of ancestrally mechanosensory organs. Consistent with this phenotype, the Pox neuro transcription factor (Poxn), which specifies chemosensory bristle identity, shows expanded expression in D. prolongata males. Poxn expression is controlled by nonadditive interactions among widely dispersed enhancers. Although some D. prolongata Poxn enhancers show increased activity, the additive component of this increase is slight, suggesting that most changes in Poxn expression are due to epistatic interactions between Poxn enhancers and trans-regulatory factors. Indeed, the expansion of D. prolongata Poxn enhancer activity is only observed in cells that express doublesex (dsx), the gene that controls sexual differentiation in Drosophila and also shows increased expression in D. prolongata males due to cis-regulatory changes. Although expanded dsx expression may contribute to increased activity of D. prolongata Poxn enhancers, this interaction is not sufficient to explain the full expansion of Poxn expression, suggesting that cis-trans interactions between Poxn, dsx, and additional unknown genes are necessary to produce the derived D. prolongata phenotype. Overall, these results demonstrate the importance of epistatic gene interactions for evolution, particularly when pivotal genes have complex regulatory architecture.
Akiyama, N., Sato, S., Tanaka, K. M., Sakai, T. and Takahashi, A. (2022). The role of the epidermis enhancer element in positive and negative transcriptional regulation of ebony in Drosophila melanogaster. G3 (Bethesda) 12(3). PubMed ID: 35100378
Summary:
The spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns in Drosophila are determined by pigments biosynthesized in the developing epidermis and the cis-regulatory elements of the genes involved in this process are well-characterized. This study reports that the known primary epidermal enhancer is dispensable for the transcriptional activation of ebony (involved in light-colored pigment synthesis) in the developing epidermis of Drosophila melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the primary epidermal enhancer by genome editing. The effect of the primary epidermal enhancer deletion on pigmentation and on the endogenous expression pattern of a mCherry-fused ebony allele was examined in the abdomen. The expression levels of the mCherry-fused ebony in the primary epidermal enhancer-deleted strains were slightly higher than that of the control strain, indicating that the sequences outside the primary epidermal enhancer have an ability to drive an expression of this gene in the epidermis. Interestingly, the primary epidermal enhancer deletion resulted in a derepression of this gene in the dorsal midline of the abdominal tergites, where dark pigmentation is present in the wild-type individuals. This indicated that the primary epidermal enhancer fragment contains a silencer. Furthermore, the endogenous expression pattern of ebony in the 2 additional strains with partially deleted primary epidermal enhancer revealed that the silencer resides within a 351-bp fragment in the 5' portion of the primary epidermal enhancer. These results demonstrated that deletion assays combined with reporter assays are highly effective in detecting the presence of positively and negatively regulating sequences within and outside the focal cis-regulatory elements.
Maguire, S. E., Afify, A., Goff, L. A. and Potter, C. J. (2022). Odorant-receptor-mediated regulation of chemosensory gene expression in the malaria mosquito Anopheles gambiae. Cell Rep 38(10): 110494. PubMed ID: 35263579
Summary:
Mosquitoes locate and approach humans based on the activity of odorant receptors (ORs) expressed on olfactory receptor neurons (ORNs). Olfactogenetic experiments in Anopheles gambiae mosquitoes revealed that the ectopic expression of an AgOR (AgOR2) in ORNs dampened the activity of the expressing neuron. This contrasts with studies in Drosophila melanogaster in which the ectopic expression of non-native ORs in ORNs confers ectopic neuronal responses without interfering with native olfactory physiology. RNA-seq analyses comparing wild-type antennae to those ectopically expressing AgOR2 in ORNs indicated that nearly all AgOR transcripts were significantly downregulated (except for AgOR2). Additional experiments suggest that AgOR2 protein rather than mRNA mediates this downregulation. Using in situ hybridization, it was found that AgOR gene choice is active into adulthood and that AgOR2 expression inhibits AgORs from turning on at this late stage. This study shows that the ORNs of Anopheles mosquitoes (in contrast to Drosophila) are sensitive to a currently unexplored mechanism of AgOR regulation.
Pascual-Garcia, P., Little, S. C. and Capelson, M. (2022). Nup98-dependent transcriptional memory is established independently of transcription. Elife 11. PubMed ID: 35289742
Summary:
Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, single-molecule RNA FISH (smFISH) was used to examine the dynamics of transcription in Drosophila cells upon repeated exposure to the steroid hormone ecdysone. smFISH was combined with mathematical modeling and it was found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously begin a slow transition into a specialized memory state characterized by a high rate of expression. Strikingly, the modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation. This prediction was confirmed experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, these findings reveal that Nup98's role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory.
Punzi, G., Ursini, G., Chen, Q., Radulescu, E., Tao, R., Huu Qi, Z., Jung, C., Bandilla, P., Ludwig, C., Heron, M., Sophie Kiesel, A., Museridze, M., Philippou-Massier, J., Nikolov, M., Renna Max Schnepf, A., Unnerstall, U., Ceolin, S., Muhlig, B., Gompel, N., Soeding, J. and Gaul, U. (2022). Large-scale analysis of Drosophila core promoter function using synthetic promoters. Mol Syst Biol 18(2): e9816. PubMed ID: 35156763
Summary:
The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it "computes" expression remains poorly understood. To dissect its function, a comprehensive structure-function analysis in was carried out in Drosophila. First, a genome-wide bioinformatic analysis was performed, providing an improved picture of the sequence motifs architecture. Then synthetic promoters' activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation) were measured, at large scale and with high accuracy using robotics and a dual luciferase reporter assay. A strong impact was observed on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans.
Secchia, S., Forneris, M., Heinen, T., Stegle, O. and Furlong, E. E. M. (2022). Simultaneous cellular and molecular phenotyping of embryonic mutants using single-cell regulatory trajectories. Dev Cell 57(4): 496-511.e498. PubMed ID: 35176234
Summary:
Developmental progression and cellular diversity are largely driven by transcription factors (TFs); yet, characterizing their loss-of-function phenotypes remains challenging and often disconnected from their underlying molecular mechanisms. This study combined single-cell regulatory genomics with loss-of-function mutants to jointly assess both cellular and molecular phenotypes. Performing sci-ATAC-seq at eight overlapping time points during Drosophila mesoderm development could reconstruct the developmental trajectories of all major muscle types and reveal the TFs and enhancers involved. To systematically assess mutant phenotypes, a single-nucleus genotyping strategy was developed to process embryo pools of mixed genotypes. Applying this to four TF mutants could identify and quantify their characterized phenotypes de novo and discover new ones, while simultaneously revealing their regulatory input and mode of action. This approach is a general framework to dissect the functional input of TFs in a systematic, unbiased manner, identifying both cellular and molecular phenotypes at a scale and resolution that has not been feasible before.

Thursday, June 16th - Evolution

Schomburg, C., Janssen, R. and Prpic, N. M. (2022). Phylogenetic analysis of forkhead transcription factors in the Panarthropoda. Dev Genes Evol 232(1): 39-48. PubMed ID: 35230523
Summary:
Fox genes encode transcription factors that contain a DNA binding domain, the forkhead domain, and are known from diverse animal species. The exact homology of the Fox genes of different species is debated and this makes inferences about the evolution of the Fox genes, and their duplications and losses difficult. We have performed phylogenetic analyses of the Fox gene complements of 32 panarthropod species. Our results confirm an ancestral complement of FoxA, FoxB, FoxC, FoxD, FoxF, FoxG, FoxJ1, FoxJ2/3, FoxK, FoxL1, FoxL2, FoxN1/4, FoxN2/3, FoxO, FoxP, and FoxQ2 in the Arthropoda, and additionally FoxH and FoxQ1 in the Panarthropoda (including tardigrades and onychophorans). We identify a novel Fox gene sub-family, that we designate as FoxT that includes two genes in Drosophila melanogaster, Circadianly Regulated Gene (Crg-1) and forkhead domain 3F (fd3F). In a very recent paper, the same new Fox gene sub-family was identified in insects (Lin, 2021). The current analysis confirms the presence of FoxT and shows that its members are present throughout Panarthropoda. The hitherto unclassified gene CG32006 from the fly Drosophila melanogaster belongs to FoxJ1. Gene losses were also detected: FoxE and FoxM were lost already in the panarthropod ancestor, whereas the loss of FoxH occurred in the arthropod ancestor. Finally, an ortholog of FoxQ1 was detected in the bark scorpion Centruroides sculpturatus, confirmed not only by phylogenetic analysis, but also by forming an evolutionarily conserved gene cluster with FoxF, FoxC, and FoxL1. This suggests that FoxQ1 belongs to the ancestral Fox gene complement in panarthropods and also in chelicerates, but has been lost at the base of the mandibulate arthropods.
Hopkins, B. R. and Perry, J. C. (2022). The evolution of sex peptide: sexual conflict, cooperation, and coevolution. Biol Rev Camb Philos Soc. PubMed ID: 35249265
Summary:
A central paradigm in evolutionary biology is that the fundamental divergence in the fitness interests of the sexes ('sexual conflict') can lead to both the evolution of sex-specific traits that reduce fitness for individuals of the opposite sex, and sexually antagonistic coevolution between the sexes. The Drosophila seminal protein Sex peptide' (SP) is perhaps the most widely cited example of a trait that appears to harm females while benefitting males. Early studies reported that the transfer of SP enhances male fitness while depressing female fitness. It is argued on three grounds that the weight of evidence does not support the view that receipt of SP decreases female fitness: (1) results from studies of SP's impact on female fitness are mixed and more often neutral or positive, with fitness costs emerging only under nutritional extremes; (2) whether costs from SP are appreciable in wild-living populations remains untested; and (3) recently described confounds in genetic manipulations of SP raise the possibility that measures of the costs and benefits of SP have been distorted. Data - from functional and evolutionary genetics and the neural circuitry of female responses to SP - suggest an evolutionary history involving the evolution of a dedicated SP-sensing apparatus in the female reproductive tract that is likely to have evolved because it benefits females, rather than harms them. Theory and evidence is explored that SP benefits females by functioning as a signal of male quality or of sperm receipt and storage (or both). The expanded view of the evolution of SP that is outlined in this study recognises the context-dependent and fluctuating roles played by both cooperative and antagonistic selection in the origin and maintenance of reproductive traits.
Chekunova, A. I., Sorokina, S. Y., Sivoplyas, E. A., Bakhtoyarov, G. N., Proshakov, P. A., Fokin, A. V., Melnikov, A. I. and Kulikov, A. M. (2021). Episodes of Rapid Recovery of the Functional Activity of the ras85D Gene in the Evolutionary History of Phylogenetically Distant Drosophila Species. Front Genet 12: 807234. PubMed ID: 35096018
Summary:
As assemblies of genomes of new species with varying degrees of relationship appear, it becomes obvious that structural rearrangements of the genome, such as inversions, translocations, and transposon movements, are an essential and often the main source of evolutionary variation. In this regard, the following questions arise. How conserved are the regulatory regions of genes? Do they have a common evolutionary origin? And how and at what rate is the functional activity of genes restored during structural changes in the promoter region? This article analyzes the evolutionary history of the formation of the regulatory region of the ras85D gene in different lineages of the genus Drosophila, as well as the participation of mobile elements in structural rearrangements and in the replacement of specific areas of the promoter region with those of independent evolutionary origin. In the process, hypotheses were substantiated about the selection of promoter elements from a number of frequently repeated motifs with different degrees of degeneracy in the ancestral sequence, as well as about the restoration of the minimum required set of regulatory sequences using a conversion mechanism or similar.
Muralidhar, P. and Veller, C. (2022). Dominance shifts increase the likelihood of soft selective sweeps. Evolution. PubMed ID: 35213740
Summary:
Genetic models of adaptation to a new environment have typically assumed that the alleles involved maintain a constant fitness dominance across the old and new environments. However, theories of dominance suggest that this should often not be the case. Instead, the alleles involved should frequently shift from recessive deleterious in the old environment to dominant beneficial in the new environment. This paper examines the consequences of these expected dominance shifts for the genetics of adaptation to a new environment. Dominance shifts were found to increase the likelihood that adaptation occurs from standing variation, and that multiple alleles from the standing variation are involved (a soft selective sweep). Furthermore, it was found that expected dominance shifts increase the haplotypic diversity of selective sweeps, rendering soft sweeps more detectable in small genomic samples. In cases where an environmental change threatens the viability of the population, it was shown that expected dominance shifts of newly beneficial alleles increase the likelihood of evolutionary rescue and the number of alleles involved. Finally, the our results are applied to a well-studied case of adaptation to a new environment: the evolution of pesticide resistance at the Ace locus in Drosophila melanogaster. Under reasonable demographic assumptions, the expected dominance shift of resistant alleles causes soft sweeps to be the most frequent outcome in this case, with the primary source of these soft sweeps being the standing variation at the onset of pesticide use, rather than recurrent mutation thereafter.
Johri, P., Stephan, W. and Jensen, J. D. (2022). Soft selective sweeps: Addressing new definitions, evaluating competing models, and interpreting empirical outliers. PLoS Genet 18(2): e1010022. PubMed ID: 35202407
Summary:
The ability to accurately identify and quantify genetic signatures associated with soft selective sweeps based on patterns of nucleotide variation has remained controversial. This study provides counter viewpoints to recent publications in PLOS Genetics that have argued not only for the statistical identifiability of soft selective sweeps, but also for their pervasive evolutionary role in both Drosophila and HIV populations. Evidence is presented that these claims owe to a lack of consideration of competing evolutionary models, unjustified interpretations of empirical outliers, as well as to new definitions of the processes themselves. The results highlight the dangers of fitting evolutionary models based on hypothesized and episodic processes without properly first considering common processes and, more generally, of the tendency in certain research areas to view pervasive positive selection as a foregone conclusion.
Rudman, S. M., Greenblum, S. I., Rajpurohit, S., Betancourt, N. J., Hanna, J., Tilk, S., Yokoyama, T., Petrov, D. A. and Schmidt, P. (2022). Direct observation of adaptive tracking on ecological time scales in Drosophila. Science 375(6586): eabj7484. PubMed ID: 35298245
Summary:
Direct observation of evolution in response to natural environmental change can resolve fundamental questions about adaptation, including its pace, temporal dynamics, and underlying phenotypic and genomic architecture. This study tracked the evolution of fitness-associated phenotypes and allele frequencies genome-wide in 10 replicate field populations of Drosophila melanogaster over 10 generations from summer to late fall. Adaptation was evident over each sampling interval (one to four generations), with exceptionally rapid phenotypic adaptation and large allele frequency shifts at many independent loci. The direction and basis of the adaptive response shifted repeatedly over time, consistent with the action of strong and rapidly fluctuating selection. Overall, clear phenotypic and genomic evidence were found of adaptive tracking occurring contemporaneously with environmental change, thus demonstrating the temporally dynamic nature of adaptation.

Wednesday June 15th - Disease Models

Shaposhnikov, M. V., Zakluta, A. S., Zemskaya, N. V., Guvatova, Z. G., Shilova, V. Y., Yakovleva, D. V., Gorbunova, A. A., Koval, L. A., Ulyasheva, N. S., Evgen'ev, M. B., Zatsepina, O. G. and Moskalev, A. A. (2022). Deletions of the cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) genes, involved in the control of hydrogen sulfide biosynthesis, significantly affect lifespan and fitness components of Drosophila melanogaster. Mech Ageing Dev 203: 111656. PubMed ID: 35247392
Summary:
The gasotransmitter hydrogen sulfide (H(2)S) is an important biological mediator, playing an essential role in many physiological and pathological processes. It is produced by transsulfuration - an evolutionarily highly conserved pathway for the metabolism of sulfur-containing amino acids methionine and cysteine. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in cysteine metabolism and H(2)S production. This study investigated the fitness components (longevity, stress resistance, viability of preimaginal stages, and reproductive function parameters) in D. melanogaster lines containing deletions of the CBS and CSE genes. Surprisingly, in most tests, CSE deletion improved, and CBS worsened the fitness. Lines with deletion of both CBS and CSE demonstrated better stress resistance and longevity than lines with single CBS deletion. At the same time, deletion of both CBS and CSE genes causes more serious disturbances of reproductive function parameters than single CBS deletion. Thus, a complex interaction of H(2)S-producing pathways and cellular stress response in determining the lifespan and fitness components of the whole organism was revealed.
Sujkowski, A., Richardson, K., Prifti, M. V., Wessells, R. J. and Todi, S. V. (2022). Endurance exercise ameliorates phenotypes in Drosophila models of spinocerebellar ataxias. Elife 11. PubMed ID: 35170431
Summary:
Endurance exercise is a potent intervention with widespread benefits proven to reduce disease incidence and impact across species. While endurance exercise supports neural plasticity, enhanced memory, and reduced neurodegeneration, less is known about the effect of chronic exercise on the progression of movement disorders such as ataxias. This study focused on three different types of ataxias, spinocerebellar ataxias type (SCAs) 2, 3, and 6, belonging to the polyglutamine (polyQ) family of neurodegenerative disorders. In Drosophila models of these SCAs, flies progressively lose motor function. This study observe marked protection of speed and endurance in exercised SCA2 flies and modest protection in exercised SCA6 models, with no benefit to SCA3 flies. Causative protein levels are reduced in SCA2 flies after chronic exercise, but not in SCA3 models, linking protein levels to exercise-based benefits. Further mechanistic investigation indicates that the exercise-inducible protein, Sestrin (Sesn), suppresses mobility decline and improves early death in SCA2 flies, even without exercise, coincident with disease protein level reduction and increased autophagic flux. These improvements partially depend on previously established functions of Sesn that reduce oxidative damage and modulate mTOR activity. This study suggests differential responses of polyQ SCAs to exercise, highlighting the potential for more extensive application of exercise-based therapies in the prevention of polyQ neurodegeneration. Defining the mechanisms by which endurance exercise suppresses polyQ SCAs will open the door for more effective treatment for these diseases.
Wei, T., Wu, L., Ji, X., Gao, Y. and Xiao, G. (2022). Ursolic Acid Protects Sodium Dodecyl Sulfate-Induced Drosophila Ulcerative Colitis Model by Inhibiting the JNK Signaling. Antioxidants (Basel) 11(2). PubMed ID: 35204308
Summary:
Ursolic acid (UA) is a bioactive molecule widely distributed in various fruits and vegetables, which was reported to play a therapeutic role in ulcerative colitis (UC) induced by toxic chemicals. However, the underlying mechanism has not been well clarified in vivo. In this study, using a Drosophila UC model induced by sodium dodecyl sulfate (SDS), the defensive effect of UA on intestinal damage was investigated. The results showed that UA could significantly protect Drosophila from the damage caused by SDS exposure. Further, UA alleviated the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by SDS and upregulated the activities of total superoxide dismutase (T-SOD) and catalase (CAT). Moreover, the proliferation and differentiation of intestine stem cells (ISCs) as well as the excessive activation of the c-Jun N-terminal kinase (JNK)-dependent JAK/STAT signaling pathway induced by SDS were restored by UA. In conclusion, UA prevents intestine injury from toxic compounds by reducing the JNK/JAK/STAT signaling pathway. UA may provide a theoretical basis for functional food or natural medicine development.
Solana-Manrique, C., Sanz, F. J., Torregrosa, I., Palomino-Schatzlein, M., Hernandez-Oliver, C., Pineda-Lucena, A. and Paricio, N. (2022). Metabolic Alterations in a Drosophila Model of Parkinson's Disease Based on DJ-1 Deficiency. Cells 11(3). PubMed ID: 35159141
Summary:
Parkinson's disease (PD) is the second-most common neurodegenerative disorder, whose physiopathology is still unclear. Moreover, there is an urgent need to discover new biomarkers and therapeutic targets to facilitate its diagnosis and treatment. Previous studies performed in PD models and samples from PD patients already demonstrated that metabolic alterations are associated with this disease. In this context, the aim of this study is to provide a better understanding of metabolic disturbances underlying PD pathogenesis. To achieve this goal, a Drosophila PD model was used based on inactivation of the DJ-1β gene (ortholog of human DJ-1). Metabolomic analyses were performed in 1-day-old and 15-day-old DJ-1β mutants and control flies using (1)H nuclear magnetic resonance spectroscopy, combined with expression and enzymatic activity assays of proteins implicated in altered pathways. The results showed that the PD model flies exhibited protein metabolism alterations, a shift from the tricarboxylic acid cycle to glycolytic pathway to obtain ATP, together with an increase in the expression of some urea cycle enzymes. Thus, these metabolic changes could contribute to PD pathogenesis and might constitute possible therapeutic targets and/or biomarkers for this disease.
Starke, E. L., Zius, K. and Barbee, S. A. (2022). FXS causing missense mutations disrupt FMRP granule formation, dynamics, and function. PLoS Genet 18(2): e1010084. PubMed ID: 35202393
Summary:
Fragile X Syndrome (FXS) is the most prevalent cause of inherited mental deficiency and is the most common monogenetic cause of autism spectral disorder (ASD). This study demonstrates that disease-causing missense mutations in the conserved K homology (KH) RNA binding domains (RBDs) of FMRP cause defects in its ability to form RNA transport granules in neurons. Using molecular, genetic, and imaging approaches in the Drosophila FXS model system, this study shows that the KH1 and KH2 domains of FMRP regulate distinct aspects of neuronal FMRP granule formation, dynamics, and transport. Furthermore, mutations in the KH domains disrupt translational repression in cells and the localization of known FMRP target mRNAs in neurons. These results suggest that the KH domains play an essential role in neuronal FMRP granule formation and function which may be linked to the molecular pathogenesis of FXS.
Sun, Z. D., Hu, J. X., Wu, J. R., Zhou, B. and Huang, Y. P. (2022). Toxicities of amyloid-beta and tau protein are reciprocally enhanced in the Drosophila model. Neural Regen Res 17(10): 2286-2292. PubMed ID: 35259851
Summary:
Extracellular aggregation of amyloid-beta (A&betaa;) and intracellular tau tangles are two major pathogenic hallmarks and critical factors of Alzheimer's disease. A linear interaction between Aβ and tau protein has been characterized in several models. Aβ induces tau hyperphosphorylation through a complex mechanism; however, the master regulators involved in this linear process are still unclear. In this study with Drosophila melanogaster, it was found that Aβ regulated tau hyperphosphorylation and toxicity by activating c-Jun N-terminal kinase. Importantly, Aβ toxicity was dependent on tau hyperphosphorylation, and flies with hypophosphorylated tau were insulated against Aβ-induced toxicity. Strikingly, tau accumulation reciprocally interfered with Aβ degradation and correlated with the reduction in mRNA expression of genes encoding Aβ-degrading enzymes, including dNep1, dNep3, dMmp2, dNep4, and dIDE. These results indicate that AAβ and tau protein work synergistically to further accelerate Alzheimer's disease progression and may be considered as a combined target for future development of Alzheimer's disease therapeutics.

Tuesday June 15th - Signaling

Mesrouze, Y., Aguilar, G., Meyerhofer, M., Bokhovchuk, F., Zimmermann, C., Fontana, P., Vissieres, A., Voshol, H., Erdmann, D., Affolter, M. and Chene, P. (2022). The role of lysine palmitoylation/myristoylation in the function of the TEAD transcription factors. Sci Rep 12(1): 4984. PubMed ID: 35322151
Summary:
The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. This report shows that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352Gln(Sd)) that is predominantly acylated on the lysine (Lys350(Sd)). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, these findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells.
Liu, J. Y., Inoshita, T., Shiba-Fukushima, K., Yoshida, S., Ogata, K., Ishihama, Y., Imai, Y. and Hattori, N. (2022). Ubiquitination at the lysine 27 residue of the parkin ubiquitin-like domain is suggestive of a new mechanism of parkin activation. Hum Mol Genet. PubMed ID: 35313349
Summary:
The mitochondrial kinase PTEN-induced kinase 1 (PINK1) and cytosolic ubiquitin ligase (E3) Parkin/PRKN are involved in mitochondrial quality control responses. PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like (Ubl) domain at serine 65 and promotes Parkin activation and translocation to damaged mitochondria. Upon Parkin activation, the Ubl domain is ubiquitinated at lysine (K) 27 and K48 residues. However, contribution of K27/K48 ubiquitination towards Parkin activity remains unclear. In this study, ubiquitination of K56 (corresponding to K27 in the human), K77 (K48 in the human), or both, was blocked by generating Drosophila Parkin (dParkin) mutants to examine the effects of Parkin Ubl domain ubiquitination on Parkin activation in Drosophila. The dParkin, in which K56 was replaced with arginine (dParkin K56R), rescued pupal lethality in flies by co-expression with PINK1, whereas dParkin K77R could not. The dParkin K56R exhibited reduced abilities of mitochondrial fragmentation and motility arrest, which are mediated by degrading Parkin E3 substrates Mitofusin and Miro, respectively. Pathogenic dParkin K56N, unlike dParkin K56R, destabilized the protein, suggesting that not only was dParkin K56N non-ubiquitin-modified at K56 but also the structure of the Ubl domain for activation was largely affected. Ubiquitin attached to K27 of the Ubl domain during PINK1-mediated Parkin activation was likely to be phosphorylated because human Parkin K27R weakened Parkin self-binding and activation in trans. Therefore, these findings suggest a new mechanism of Parkin activation, where an activation complex is formed through phospho-ubiquitin attachment on the K27 residue of the Parkin Ubl domain.
Roberto, N., Becam, I., Plessis, A. and Holmgren, R. A. (2022). Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels. Development 149(6). PubMed ID: 35290435
Summary:
Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. This study shows that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, evidence is provided that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill.
Texada, M. J., Lassen, M., Pedersen, L. H., Koyama, T., Malita, A. and Rewitz, K. (2022). Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila. Curr Biol 32(7): 1548-1562. PubMed ID: 35245460
Summary:
This study shows that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty.
Wang, G., Zhai, C., Ji, X., Wang, E., Zhao, S., Qian, C., Yu, D., Wang, Y. and Wu, S. (2022). C-terminal-mediated homodimerization of Expanded is critical for its ability to promote Hippo signalling in Drosophila. FEBS Lett. PubMed ID: 35278215
Summary:
Hippo signalling plays key role in tissue growth and homeostasis, and its dysregulation is implicated in various human diseases. Expanded (Ex) is an important upstream activator of Hippo signalling; however, how Ex activates Hippo signalling is still poorly understood. This study demonstrate that Ex forms a homodimer via C-terminal interaction, and that the ExC2 region (912-1164 aa) is sufficient and essential for Ex dimerization. Functional analysis shows that ExC2 is required for Ex to promote the phosphorylation and inactivation of Yki in Drosophila cells. Further in vivo analysis shows that ExC2 is important for Ex to control Drosophila tissue growth. This study thus, uncovers a novel mechanism whereby Ex homodimerization mediates its full activation to promote Hippo signalling in growth control.
Na, H. J., Abramowitz, L. K. and Hanover, J. A. (2022). Cytosolic O-GlcNAcylation and PNG1 maintain Drosophila gut homeostasis by regulating proliferation and apoptosis. PLoS Genet 18(3): e1010128. PubMed ID: 35294432
Summary:
It remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. This study utilized the Drosophila gut and uncover a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis.

Monday, June 13th - Immune response

Aalto, A., Martinez-Chacon, G., Kietz, C., Tsyganova, N., Kreutzer, J., Kallio, P., Broemer, M. and Meinander, A. (2022). M1-linked ubiquitination facilitates NF-κB activation and survival during sterile inflammation. Febs j. PubMed ID: 35263507
Summary:
Methionine 1 (M1)-linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor-κB (NF-κB) signalling and is important for clearance of pathogen infection in Drosophila melanogaster. M1-linked ubiquitin (M1-Ub) chains are assembled by the linear ubiquitin E3 ligase (LUBEL) in flies. The role of LUBEL was studied in sterile inflammation induced by different types of cellular stresses. The LUBEL was found to catalyse formation of M1-Ub chains in response to hypoxic, oxidative and mechanical stress conditions. LUBEL is shown to be important for flies to survive low oxygen conditions and paraquat-induced oxidative stress. This protective action seems to be driven by stress-induced activation of the NF-κB transcription factor Relish via the immune deficiency (Imd) pathway. In addition to LUBEL, the intracellular mediators of Relish activation, including the transforming growth factor activating kinase 1 (Tak1), Drosophila inhibitor of apoptosis (IAP) Diap2, the IκB kinase γ (IKKγ) Kenny and the initiator caspase Death-related ced-3/Nedd2-like protein (Dredd), but not the membrane receptor peptidoglycan recognition protein (PGRP)-LC, are shown to be required for sterile inflammatory response and survival. Finally, it was shown that the stress-induced upregulation of M1-Ub chains in response to hypoxia, oxidative and mechanical stress is also induced in mammalian cells and protects from stress-induced cell death. Taken together, these results suggest that M1-Ub chains are important for NF-κB signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer.
Soory, A. and Ratnaparkhi, G. S. (2022). SUMOylation of Jun fine-tunes the Drosophila gut immune response. PLoS Pathog 18(3): e1010356. PubMed ID: 35255103
Summary:
Post-translational modification by the small ubiquitin-like modifier, SUMO can modulate the activity of its conjugated proteins in a plethora of cellular contexts. The effect of SUMO conjugation of proteins during an immune response is poorly understood in Drosophila. Previous work found that the transcription factor Jra, the Drosophila Jun ortholog and a member of the AP-1 complex is one such SUMO target. This study found that Jra is a regulator of the Pseudomonas entomophila induced gut immune gene regulatory network, modulating the expression of a few thousand genes, as measured by quantitative RNA sequencing. Decrease in Jra in gut enterocytes is protective, suggesting that reduction of Jra signaling favors the host over the pathogen. In Jra, lysines 29 and 190 are SUMO conjugation targets, with the JraK29R+K190R double mutant being SUMO conjugation resistant (SCR). Interestingly, a JraSCR fly line, generated by CRISPR/Cas9 based genome editing, is more sensitive to infection, with adults showing a weakened host response and increased proliferation of Pseudomonas. Transcriptome analysis of the guts of JraSCR and JraWT flies suggests that lack of SUMOylation of Jra significantly changes core elements of the immune gene regulatory network, which include antimicrobial agents, secreted ligands, feedback regulators, and transcription factors. Mechanistically, SUMOylation attenuates Jra activity, with the TFs, forkhead, anterior open, activating transcription factor 3 and the master immune regulator Relish being important transcriptional targets. This study implicates Jra as a major immune regulator, with dynamic SUMO conjugation/deconjugation of Jra modulating the kinetics of the gut immune response.
Mongelli, V., Lequime, S., Kousathanas, A., Gausson, V., Blanc, H., Nigg, J., Quintana-Murci, L., Elena, S. F. and Saleh, M. C. (2022). Innate immune pathways act synergistically to constrain RNA virus evolution in Drosophila melanogaster. Nat Ecol Evol. PubMed ID: 35273366
Summary:
Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. This evolutionary arms-race was study, and the impact of the innate immune system on viral population diversity and evolution was assessed using Drosophila melanogaster as model host and its natural pathogen Drosophila C virus (DCV). Eight fly genotypes were isogenized, generating animals defective for RNAi, Imd and Toll innate immune pathways as well as pathogen-sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected with DCV and the virus was serially passaged ten times via reinfection in naive flies. Viral population diversity was studied after each viral passage by high-throughput sequencing and infection phenotypes were assessed at the beginning and at the end of the evolution experiment. The absence of any of the various immune pathways studied increased viral genetic diversity while attenuating virulence. Strikingly, these effects were observed in a range of host factors described as having mainly antiviral or antibacterial functions. Together, these results indicate that the innate immune system as a whole and not specific antiviral defence pathways in isolation, generally constrains viral diversity and evolution.
Zhou, H., Li, S., Pan, W., Wu, S., Ma, F. and Jin, P. (2022). Interaction of lncRNA-CR33942 with Dif/Dorsal Facilitates Antimicrobial Peptide Transcriptions and Enhances Drosophila Toll Immune Responses. J Immunol 208(8): 1978-1988. PubMed ID: 35379744
Summary:
The Drosophila Toll signaling pathway mainly responds to Gram-positive (G(+)) bacteria or fungal infection, which is highly conserved with mammalian TLR signaling pathway. Although many positive and negative regulators involved in the immune response of the Toll pathway have been identified in Drosophila, the roles of long noncoding RNAs (lncRNAs) in Drosophila Toll immune responses are poorly understood to date. In this study, the results demonstrate that lncRNA-CR33942 is mainly expressed in the nucleus and upregulated after Micrococcus luteus infection. Especially, lncRNA-CR33942 not only modulates differential expressions of multiple antimicrobial peptide genes but also affects the Drosophila survival rate during response to G(+) bacterial infection based on the transiently overexpressing and the knockdown lncRNA-CR33942 assays in vivo. Mechanically, lncRNA-CR33942 interacts with the NF-κB transcription factors Dorsal-related immunity factor/Dorsal to promote the transcriptions of antimicrobial peptides drosomycin and metchnikowin, thus enhancing Drosophila Toll immune responses. Taken together, this study identifies lncRNA-CR33942 as a positive regulator of Drosophila innate immune response to G(+) bacterial infection to facilitate Toll signaling via interacting with Dorsal-related immunity factor/Dorsal. It would be helpful to reveal the roles of lncRNAs in Toll immune response in Drosophila and provide insights into animal innate immunity.
Evans, C. J., Liu, T., Girard, J. R. and Banerjee, U. (2022). Injury-induced inflammatory signaling and hematopoiesis in Drosophila. Proc Natl Acad Sci U S A 119(12): e2119109119. PubMed ID: 35286208
Summary:
This study explores mechanisms by which stress caused by acute injury affects blood cell development and inflammatory response in Drosophila. Similar to their mammalian myeloid counterparts, these cells are predisposed to sense and react to sterile injury at distant sites. Upon sterile injury, a breach of epidermis sets up a reactive oxygen species-based signal that bypasses the pathogen-sensing apparatus of septic immune challenge, but merges downstream to activate Toll. A number of autonomous and nonautonomous signaling pathways follow in a sequence and are mapped temporally by the appearance of their corresponding molecular phenotypes. A cell-type that fights deposited parasitic wasp eggs appears with sterile injury without the immune challenge, perhaps in anticipation, because in nature injury is usually followed by infection.
Glittenberg, M., Kounatidis, I., Atilano, M. and Ligoxygakis, P. (2022). A genetic screen in Drosophila reveals the role of fucosylation in host susceptibility to Candida infection. Dis Model Mech. PubMed ID: 35142345
Summary:
Candida infections constitute a blind spot in global public health as very few new anti-fungal drugs are being developed. Genetic surveys of host susceptibilities to such infections using mammalian models have certain disadvantages in that obtaining results is time-consuming owing to relatively long lifespans and these results have low statistical resolution because sample sizes are usually small. This paper reports a targeted genetic screening of 5698 RNAi lines encompassing 4135 Drosophila genes with human homologues, several of which were identified as important for host survival after Candida albicans infection. These include genes in a variety of functional classes encompassing gene expression, intracellular signalling, metabolism, and enzymatic regulation. Analysis of one of the screen hits, the infection-induced α-(1,3)-fucosylase FucTA, showed that N-glycan fucosylation has several targets among proteins involved in host defence supplying multiple avenues of investigation for the mechanistic analysis of host survival to systemic C. albicans infection.

Friday, June 10th - Apoptosis and Autophagy

Murakawa, T., Nakamura, T., Kawaguchi, K., Murayama, F., Zhao, N., Stasevich, T. J., Kimura, H. and Fujita, N. (2022). A Drosophila toolkit for HA-tagged proteins unveils a block in autophagy flux in the last instar larval fat body. Development 149(6). PubMed ID: 35319746
Summary:
For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with a POI that harbors different tags are needed but generation of these strains is still labor-intensive work. To overcome this, a versatile Drosophila toolkit was developed with a genetically encoded single-chain variable fragment for the HA epitope tag: 'HA Frankenbody'. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Mechanistically, lysosomal activity was downregulated at this stage, and endocytosis, but not autophagy, was indispensable for the swelling of lysosomes. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, it is proposed that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.
Bhattacharjee, A., Urmosi, A., Jipa, A., Kovacs, L., Deak, P., Szabo, A. and Juhasz, G. (2022). Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses. Autophagy: 1-12. PubMed ID: 35184662
Summary:
SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Using a genetically non-redundant Drosophila model, this study shows that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. Accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response.
Kakanj, P., Bhide, S., Moussian, B. and Leptin, M. (2022). Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia. Embo j: e109992. PubMed ID: 35262206
Summary:
Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. This study shows that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo-membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub-cellular membranes, as shown by its suppression of experimentally induced laminopathy-like nuclear defects. These findings reveal a function for TORC1-mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing.
Tsapras, P. and Nezis, I. P. (2022). A yeast two-hybrid screening identifies novel Atg8a interactors in Drosophila. Autophagy: 1-2. PubMed ID: 35226578
Summary:
Macroautophagy/autophagy-related protein Atg8/LC3 is important for autophagosome biogenesis and required for selective degradation of various substrates. In a recent study, a yeast two-hybrid screening was performed to identify proteins that interact with Atg8a, the Drosophila homolog of Atg8/LC3. The screening identified several Atg8a-interacting proteins. These proteins include: i) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, ref(2)P and key (Kenny); ii) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4; and iii) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1. We showed that Tak1, as well as its co-activator, Tab2, both interact with Atg8a and are substrates for selective autophagic clearance. It was also determined that SH3PX1 interacts with Tab2 and is necessary for the effective regulation of the immune-deficiency (IMD) pathway. These findings suggest a mechanism for the regulatory interactions between Tak1-Tab2-SH3PX1 and Atg8a, which contribute to the fine-tuning of the IMD pathway.
Guo, X., Li, Z., Zhu, X., Zhan, M., Wu, C., Ding, X., Peng, K., Li, W., Ma, X., Lv, Z., Lu, L. and Xue, L. (2022). A coherent FOXO3-SNAI2 feed-forward loop in autophagy. Proc Natl Acad Sci U S A 119(11): e2118285119. PubMed ID: 35271390
Summary:
Understanding autophagy regulation is instrumental in developing therapeutic interventions for autophagy-associated disease. This study identified SNAI2 as a regulator of autophagy from a genome-wide screen in HeLa cells. Upon energy stress, SNAI2 is transcriptionally activated by FOXO3 and interacts with FOXO3 to form a feed-forward regulatory loop to reinforce the expression of autophagy genes. Of note, SNAI2-increased FOXO3-DNA binding abrogates CRM1-dependent FOXO3 nuclear export, illuminating a pivotal role of DNA in the nuclear retention of nucleocytoplasmic shuttling proteins. Moreover, a dFoxO-Snail feed-forward loop regulates both autophagy and cell size in Drosophila, suggesting this evolutionarily conserved regulatory loop is engaged in more physiological activities.
Raymond, M. H., Davidson, A. J., Shen, Y., Tudor, D. R., Lucas, C. D., Morioka, S., Perry, J. S. A., Krapivkina, J., Perrais, D., Schumacher, L. J., Campbell, R. E., Wood, W. and Ravichandran, K. S. (2022). . Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo. Science 375(6585): 1182-1187. PubMed ID: 35271315
Summary:
Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. This study developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, multiple qualitative and quantitative features were uncovered of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.

Thursday, June 9th - Cytoskeleton and Junctions

Sherrard, K. M., Cetera, M. and Horne-Badovinac, S. (2021). DAAM mediates the assembly of long-lived, treadmilling stress fibers in collectively migrating epithelial cells in Drosophila. Elife 10. PubMed ID: 34812144
Summary:
Stress fibers (SFs) are actomyosin bundles commonly found in individually migrating cells in culture. However, whether and how cells use SFs to migrate in vivo or collectively is largely unknown. Studying the collective migration of the follicular epithelial cells in Drosophila, it was found that the SFs in these cells show a novel treadmilling behavior that allows them to persist as the cells migrate over multiple cell lengths. Treadmilling SFs grow at their fronts by adding new integrin-based adhesions and actomyosin segments over time. This causes the SFs to have many internal adhesions along their lengths, instead of adhesions only at the ends. The front-forming adhesions remain stationary relative to the substrate and typically disassemble as the cell rear approaches. By contrast, a different type of adhesion forms at the SF's terminus that slides with the cell's trailing edge as the actomyosin ahead of it shortens. This study further showed that SF treadmilling depends on cell movement and identify a developmental switch in the formins that mediate SF assembly, with Dishevelled-associated activator of morphogenesis acting during migratory stages and Diaphanous acting during postmigratory stages. It is proposed that treadmilling SFs keep each cell on a linear trajectory, thereby promoting the collective motility required for epithelial migration.
Lechuga, S., Cartagena-Rivera, A. X., Khan, A., Crawford, B. I., Narayanan, V., Conway, D. E., Lehtimaki, J., Lappalainen, P., Rieder, F., Longworth, M. S. and Ivanov, A. I. (2022). A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair. Faseb j 36(5): e22290. PubMed ID: 35344227
Summary:
Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. This study found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. These findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.
Lu, W., Lakonishok, M., Serpinskaya, A. S. and Gelfand, V. I. (2022). A novel mechanism of bulk cytoplasmic transport by cortical dynein in Drosophila ovary. Elife 11. PubMed ID: 35170428
Summary:
Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. Previous work has shown that cytoplasmic dynein is required for Drosophila oocyte growth and has assumed that it simply transports cargoes along microtubule tracks from nurse cells to the oocyte. This study reports that instead of transporting individual cargoes along stationary microtubules into the oocyte, cortical dynein actively moves microtubules within nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. This robust microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein performs bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. It is proposed that the dynein-driven microtubule flow could serve as a novel mode of fast cytoplasmic transport.
Trujillo, A. S., Hsu, K. H., Viswanathan, M. C., Cammarato, A. and Bernstein, S. I. (2022). The R369 Myosin Residue within Loop 4 Is Critical for Actin Binding and Muscle Function in Drosophila. Int J Mol Sci 23(5). PubMed ID: 35269675
Summary:
The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin's upper 50 kDa subdomain. To probe the importance of R369, this study introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, these findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism.
Selvaggi, L., Ackermann, M., Pasakarnis, L., Brunner, D. and Aegerter, C. M. (2021). Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure. Biophys J. PubMed ID: 34971619
Summary:
The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. A patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. This tool was used to quantify the in vivo force production of Myosin II waves that are observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing quantitative values on an active Myosin-driven force, this study has elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and the mechanical properties of the dorsal yolk cell surface were characterized.
Kulshammer, E., Kilinc, M., Csordas, G., Bresser, T., Nolte, H. and Uhlirova, M. (2022). The mechanosensor Filamin A/Cheerio promotes tumourigenesis via specific interactions with components of the cell cortex. Febs j. PubMed ID: 35191183
Summary:
Cancer development has been linked to aberrant sensing and interpretation of mechanical cues and force-generating properties. This study shows that upregulation of the actin crosslinking protein Cheerio (Cher), the fly ortholog of Filamin A (FLNA), and the conformation of its mechanosensitive region (MSR) are instrumental to the malignancy of polarity-deficient, Ras-driven tumours in Drosophila epithelia. This study shows that impaired growth and cytoskeletal contractility of tumours devoid of cher can be rescued by stimulating myosin activity. Profiling the Cher interactome in tumour-bearing imaginal discs identified several components of the cell cortex, including the β-heavy Spectrin Karst (Kst), the scaffolding protein Big bang (Bbg), and 14-3-3ε. Cher binds Bbg through the MSR while the interaction with 14-3-3ε and Kst is MSR-independent. Importantly, these genetic studies define Bbg, Kst, and 14-3-3ε as tumour suppressors. The tumour-promoting function of Cher thus relies on its capacity to control the contractile state of the cytoskeleton through interactions with myosin and specific components of the cell cortex.

Wednesday, June 8th - Embryonic Development

Stern, T., Shvartsman, S. Y. and Wieschaus, E. F. (2022). Deconstructing gastrulation at single-cell resolution. Curr Biol. PubMed ID: 35290798
Summary:
Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets, which are driven by cell divisions, cell shape changes, and cell intercalations. Each of these behaviors has been associated with distinct aspects of gastrulation and has been a subject of intense research using genetic, cell biological, and more recently, biophysical approaches. Most of these studies, however, focus either on cellular processes driving gastrulation or on large-scale tissue deformations. Recent advances in microscopy and image processing create a unique opportunity for integrating these complementary viewpoints. This study takes a step toward bridging these complementary strategies and deconstruct the early stages of gastrulation in the entire Drosophila embryo. The approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. The detected events are then mapped back onto the blastoderm shell, providing an intuitive visual means to examine complex cellular activity patterns within the context of their initial anatomic domains. By analyzing these maps, it was identified that the loss of nearly half of surface cells to invaginations is compensated primarily by transient mitotic rounding. In addition, by analyzing mapped cell intercalation events, direct quantitative relations between intercalation frequency and the rate of axis elongation were derived. This work is setting the stage for systems-level dissection of a pivotal step in animal development.
Guo, H., Swan, M. and He, B. (2022). Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination. Elife 11. PubMed ID: 35195065
Summary:
Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, it was find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. It is proposed that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.
Fuentes, M. A. and He, B (2022). The cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination. Development 149(6). PubMed ID: 35302584
Summary:
Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, an important role was identified for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. Depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. These findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.
Singh, A. P., Wu, P., Ryabichko, S., Raimundo, J., Swan, M., Wieschaus, E., Gregor, T. and Toettcher, J. E. (2022). Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation. Cell Rep 38(12): 110543. PubMed ID: 35320726
Summary:
Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. This study shows that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor in vivo. Light-controlled versions of the Bicoid transcription factor were generated and their effects on downstream gap genes was studied in embryos. The results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, it was found that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirpsggmj transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.
Ferree, P. L., Xing, M., Zhang, J. Q. and Di Talia, S. (2022). Structure-function analysis of Cdc25(Twine) degradation at the Drosophila maternal-to-zygotic transition. Fly (Austin) 16(1): 111-117. PubMed ID: 35227166
Summary:
Downregulation of protein phosphatase Cdc25(Twine) activity is linked to remodelling of the cell cycle during the Drosophila maternal-to-zygotic transition (MZT). This study presents a structure-function analysis of Cdc25(Twine). Chimeras were used to show that the N-terminus regions of Cdc25(Twine) and Cdc25(String) control their differential degradation dynamics. Deletion of different regions of Cdc25(Twine) reveals a putative domain involved in and required for its rapid degradation during the MZT. Notably, a very similar domain is present in Cdc25(String) and deletion of the DNA replication checkpoint results in similar dynamics of degradation of both Cdc25(String) and Cdc25(Twine). Finally, this study shows that Cdc25(Twine) degradation is delayed in embryos lacking the left arm of chromosome III. Thus, a model is proposed for the differential regulation of Cdc25 at the Drosophila MZT.
Loganathan, R., Levings, D. C., Kim, J. H., Wells, M. B., Chiu, H., Wu, Y., Slattery, M. and Andrew, D. J. (2022). Ribbon boosts ribosomal protein gene expression to coordinate organ form and function. J Cell Biol 221(4). PubMed ID: 35195669
Summary:
Cell growth is well defined for late (postembryonic) stages of development, but evidence for early (embryonic) cell growth during postmitotic morphogenesis is limited. This study reports early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG) and trachea. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence specific, suggesting that specificity is achieved through cofactor interactions. Accordingly, Rib's ability to physically interact with each of the three known regulators of RPG transcription was demonstrated. Surprisingly, Rib-dependent early cell growth in another tubular organ, the embryonic trachea, is not mediated by direct RPG transcription. These findings support a model of early cell growth customized by transcriptional regulatory networks to coordinate organ form and function.

Tuesday, June 7th - Adult physiology

Shukla, N. and Kolthur-Seetharam, U. (2022). Drosophila Sirtuin 6 mediates developmental diet-dependent programming of adult physiology and survival. Aging Cell 21(3): e13576. PubMed ID: 35233942
Summary:
Organisms in the wild experience unpredictable and diverse food availability throughout their lifespan. Over-/under-nutrition during development and in adulthood is known to dictate organismal survival and fitness. Studies using model systems have also established long-term effects of developmental dietary alterations on life-history traits. However, the underlining genetic/molecular factors, which differentially couple nutrient inputs during development with fitness later in life are far less understood. Using Drosophila and loss/gain of function perturbations, serendipitous findings demonstrate an essential role of Sirtuin 6 in regulating larval developmental kinetics, in a nutrient-dependent manner. The absence of Sirt6 affected ecdysone and insulin signalling and led to accelerated larval development. Moreover, varying dietary glucose and yeast during larval stages resulted in enhanced susceptibility to metabolic and oxidative stress in adults. This study also demonstrated an evolutionarily conserved role for Sirt6 in regulating physiological homeostasis, physical activity and organismal lifespan, known only in mammals until now. These results highlight gene-diet interactions that dictate thresholding of nutrient inputs and physiological plasticity, operative across development and adulthood. In summary, besides showing its role in invertebrate ageing, this study also identifies Sirt6 as a key factor that programs macronutrient-dependent life-history traits.
Schellinger, J. N., Sun, Q., Pleinis, J. M., An, S. W., Hu, J., Mercenne, G., Titos, I., Huang, C. L., Rothenfluh, A. and Rodan, A. R. (2022). Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade. Curr Biol 32(6): 1429-1438. PubMed ID: 35303418
Summary:
Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies. Whether and how intracellular chloride regulates circadian rhythms remains controversial. This study demonstrates a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLN(v)) pacemaker neurons. In control flies, intracellular chloride increases in sLN(v)s over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLN(v) pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.
Meiselman, M. R., Alpert, M. H., Cui, X., Shea, J., Gregg, I., Gallio, M. and Yapici, N. (2022). Recovery from cold-induced reproductive dormancy is regulated by temperature-dependent AstC signaling. Curr Biol 32(6): 1362-1375. PubMed ID: 35176227
Summary:
Animals have evolved a variety of behaviors to cope with adverse environmental conditions. Similar to other insects, the fly, Drosophila melanogaster, responds to sustained cold by reducing its metabolic rate and arresting its reproduction. This study shows that a subset of dorsal neurons (DN3s) that express the neuropeptide allatostatin C (AstC) facilitates recovery from cold-induced reproductive dormancy. The activity of AstC-expressing DN3s, as well as AstC peptide levels, are suppressed by cold. Cold temperature also impacts AstC levels in other Drosophila species and mosquitoes, Aedes aegypti, and Anopheles stephensi. The stimulatory effect of AstC on egg production is mediated by cholinergic AstC-R2 neurons. These results demonstrate that DN3s coordinate female reproductive capacity with environmental temperature via AstC signaling. AstC/AstC-R2 is conserved across many insect species and their role in regulating female reproductive capacity makes them an ideal target for controlling the population of agricultural pests and human disease vectors.
Koppik, M. and Fricke, C. (2022). Sex peptide receipt alters macronutrient utilization but not optimal yeast-sugar ratio in Drosophila melanogaster females. J Insect Physiol 139: 104382. PubMed ID: 35318041
Summary:
Upon mating, females alter a multitude of physiological and morphological traits to accommodate the demands of reproduction. Changes not only include reproductive tissues but also non-reproductive tissues. For example, in Drosophila melanogaster the gut increases in circumference after mating, likely to facilitate a higher absorption and provision of macronutrients to maturing eggs. A male ejaculatory protein, the sex peptide, is instrumental to mediating several post-mating changes and receipt increases nutrient uptake as well as shifts taste preferences in mated females. This study tested whether sex peptide receipt also alters the protein: carbohydrate ratio at which females maximize their fitness. To test this, females were mated to males lacking sex peptide or control males and fed them with known volumes and concentrations of sugar and yeast. This enabled determination of how the sugar to yeast ratio affects lifetime egg output as well as lifespan of females mated to the two male types. Sex peptide did not shift the optimal ratio. Instead, sex peptide receipt aided females in increasing their egg output at low macronutrient concentrations, but this advantage disappeared at higher macronutrient intake rates. Assuming that nutrient limitation might be common, then receipt of SP is beneficial under poor conditions.
Millington, J. W., Biswas, P., Chao, C., Xia, Y. H., Wat, L. W., Brownrigg, G. P., Sun, Z., Basner-Collins, P. J., Klein Geltink, R. I. and Rideout, E. J. (2022). A low-sugar diet enhances Drosophila body size in males and females via sex-specific mechanisms. Development 149(6). PubMed ID: 35195254
Summary:
In Drosophila, changes to dietary protein elicit different body size responses between the sexes. Whether these differential body size effects extend to other macronutrients remains unclear. This study shows that lowering dietary sugar (0S diet) enhanced body size in male and female larvae. Despite an equivalent phenotypic effect between the sexes,sex-specific changes were detected to signalling pathways, transcription and whole-body glycogen and protein. In males, the low-sugar diet augmented insulin/insulin-like growth factor signalling pathway (IIS) activity by increasing insulin sensitivity, where increased IIS was required for male metabolic and body size responses in 0S. In females reared on low sugar, IIS activity and insulin sensitivity were unaffected, and IIS function did not fully account for metabolic and body size responses. Instead, a female-biased requirement for the Target of rapamycin pathway was detected in regulating metabolic and body size responses. Together, these data suggest the mechanisms underlying the low-sugar-induced increase in body size are not fully shared between the sexes, highlighting the importance of including males and females in larval studies even when similar phenotypic outcomes are observed.
Murakami, A., Nagao, K., Sakaguchi, R., Kida, K., Hara, Y., Mori, Y., Okabe, K., Harada, Y. and Umeda, M. (2022). Cell-autonomous control of intracellular temperature by unsaturation of phospholipid acyl chains. Cell Rep 38(11): 110487. PubMed ID: 35294880
Summary:
Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, this study found that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F(1)F(o)-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. This study also revealed that F(1)F(o)-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, a cell-autonomous mechanism is proposed for intracellular temperature control during environmental temperature changes.

Monday, June 6 - Adult neural development and function

Sears, J. C. and Broadie, K. (2022). Temporally and Spatially Localized PKA Activity within Learning and Memory Circuitry Regulated by Network Feedback. eNeuro 9(2). PubMed ID: 35301221
Summary:
Dynamic functional connectivity within brain circuits requires coordination of intercellular signaling and intracellular signal transduction. Critical roles for cAMP-dependent protein kinase A (PKA) signaling are well established in the Drosophila mushroom body (MB) learning and memory circuitry, but local PKA activity within this well-mapped neuronal network is uncharacterized. This study use an in vivo PKA activity sensor (PKA-SPARK) to test spatiotemporal regulatory requirements in the MB axon lobes. Immature animals had little detectable PKA activity, whereas postcritical period adults showed high field-selective activation primarily in just 3/16 defined output regions. In addition to the age-dependent PKA activity in distinct α'/β' lobe nodes, females show sex-dependent elevation compared with males in these same restricted regions. Loss of neural cell body Fragile X mental retardation protein (FMRP) and Rugose [human Neurobeachin (NBEA)] suppresses localized PKA activity, whereas overexpression (OE) of MB lobe PKA-synergist Meng-Po (human SBK1) promotes PKA activity. Elevated Meng-Po subverts the PKA age-dependence, with elevated activity in immature animals, and spatial-restriction, with striking γ lobe activity. Testing circuit signaling requirements with temperature-sensitive shibire (human Dynamin) blockade, broadly expanded PKA activity was found within the MB lobes. Using transgenic tetanus toxin to block MB synaptic output, greatly heightened PKA activity was found in virtually all MB lobe fields, although the age-dependence is maintained. It is concluded spatiotemporally restricted PKA activity signaling within this well-mapped learning/memory circuit is age-dependent and sex-dependent, driven by FMRP-Rugose pathway activation, temporally promoted by Meng-Po kinase function, and restricted by output neurotransmission providing network feedback.
Dubal, D., Moghe, P., Verma, R. K., Uttekar, B. and Rikhy, R. (2022). Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila. PLoS Genet 18(2): e1010055. PubMed ID: 35157701
Summary:
Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. This study assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. These results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.
Silva, B., Mantha, O. L., Schor, J., Pascual, A., Placais, P. Y., Pavlowsky, A. and Preat, T. (2022). Glia fuel neurons with locally synthesized ketone bodies to sustain memory under starvation. Nat Metab 4(2): 213-224. PubMed ID: 35177854
Summary:
\During starvation, mammalian brains can adapt their metabolism, switching from glucose to alternative peripheral fuel sources. In the Drosophila starved brain, memory formation is subject to adaptative plasticity, but whether this adaptive plasticity relies on metabolic adaptation remains unclear. This study shows that during starvation, neurons of the fly olfactory memory centre import and use ketone bodies (KBs) as an energy substrate to sustain aversive memory formation. Local providers within the brain, the cortex glia, were identified that use their own lipid store to synthesize KBs before exporting them to neurons via monocarboxylate transporters. Finally, it was show that the master energy sensor AMP-activated protein kinase regulates both lipid mobilization and KB export in cortex glia. These data provide a general schema of the metabolic interactions within the brain to support memory when glucose is scarce.
Miozzo, F., Valencia-Alarcon, E. P., Stickley, L., Majcin Dorcikova, M., Petrelli, F., Tas, D., Loncle, N., Nikonenko, I., Bou Dib, P. and Nagoshi, E. (2022). Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor. Nat Commun 13(1): 1426. PubMed ID: 35301315
Summary:
Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, this study demonstrated that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. It was further shown that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. These results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.
O'Neill, R. S. and Rusan, N. M. (2022). Traip controls mushroom body size by suppressing mitotic defects. Development 149(7). PubMed ID: 35297981
Summary:
Microcephaly is a failure to develop proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple functions in DDR. We characterized the Drosophila TRAIP homolog nopo, hereafter traip, and found that traip mutants (traip-) have a brain-specific defect in the mushroom body (MB). traip- MBs were smaller and contained fewer neurons, but no neurodegeneration, consistent with human primary microcephaly. Reduced neuron numbers in traip- were explained by premature loss of MB neuroblasts (MB-NBs), in part via caspase-dependent cell death. Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy or micronuclei. Traip localization during mitosis is sufficient for MB development, suggesting that Traip can repair chromosome bridges during mitosis if necessary. The results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and premature neural stem cell loss to promote proper neuron number.
Robinson, B. S., Norman-Tenazas, R., Cervantes, M., Symonette, D., Johnson, E. C., Joyce, J., Rivlin, P. K., Hwang, G. M., Zhang, K. and Gray-Roncal, W. (2022). Online learning for orientation estimation during translation in an insect ring attractor network. Sci Rep 12(1): 3210. PubMed ID: 35217679
Summary:
Insect neural systems are a promising source of inspiration for new navigation algorithms, especially on low size, weight, and power platforms. There have been unprecedented recent neuroscience breakthroughs with Drosophila in behavioral and neural imaging experiments as well as the mapping of detailed connectivity of neural structures. General mechanisms for learning orientation in the central complex (CX) of Drosophila have been investigated previously; however, it is unclear how these underlying mechanisms extend to cases where there is translation through an environment (beyond only rotation), which is critical for navigation in robotic systems. This study developed a CX neural connectivity-constrained model that performs sensor fusion, as well as unsupervised learning of visual features for path integration; the viability of this circuit was demonstrated for use in robotic systems in simulated and physical environments. Furthermore, a theoretical understanding is demonstrated of how distributed online unsupervised network weight modification can be leveraged for learning in a trajectory through an environment by minimizing orientation estimation error. Overall, these results may enable a new class of CX-derived low power robotic navigation algorithms and lead to testable predictions to inform future neuroscience experiments.

Friday, June 3rd - Gonads

Patlar, B. and Civetta, A. (2022). Seminal fluid gene expression and reproductive fitness in Drosophila melanogaster. BMC Ecol Evol 22(1): 20. PubMed ID: 35196983
Summary:
The rapid evolution of seminal fluid proteins (SFPs) has been suggested to be driven by adaptations to postcopulatory sexual selection (e.g. sperm competition). However, it has been recently shown that most SFPs evolve rapidly under relaxed selective pressures. Given the role of SFPs in competition for fertilization phenotypes, like the ability to transfer and store sperm and the modulation of female receptivity and ovulation, the prevalence of selectively relaxed SFPs appears as a conundrum. One possible explanation is that selection on SFPs might be relaxed in terms of protein amino acid content, but adjustments of expression are essential for post-mating function. Interestingly, there is a general lack of systematic implementation of gene expression perturbation assays to monitor their effect on phenotypes related to sperm competition. This study successfully manipulated the expression of 16 SFP encoding genes using tissue-specific knockdowns (KDs) and determined the effect of these genes' perturbation on three important post-mating phenotypes: female refractoriness to remating, defensive (P1), and offensive (P2) sperm competitive abilities in Drosophila melanogaster. These analyses show that KDs of tested SFP genes do not affect female refractoriness to remating and P2, however, most gene KDs significantly decreased P1. Moreover, KDs of SFP genes that are selectively constrained in terms of protein-coding sequence evolution have lower P1 than KDs of genes evolving under relaxed selection. These results suggest a more predominant role, than previously acknowledged, of variation in gene expression than coding sequence changes on sperm competitive ability in D. melanogaster.
Mao, B., Zhang, W., Zheng, Y., Li, D., Chen, M. Y. and Wang, Y. F. (2022). Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster. Insect Sci. PubMed ID: 35271765
Summary:
The most common phenotype induced by the endosymbiont Wolbachia in insects is cytoplasmic incompatibility, where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females. This suggests that some modifications are induced in host sperms during spermatogenesis by Wolbachia. To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster, this study applied isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic strategy combined with titanium dioxide (TiO(2)) enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male reproductive systems in D. melanogaster. 182 phosphopeptides were identified, defining 140 phosphoproteins, that have at least a 1.2 fold change in abundance with a P-value of <0.05. Most of the differentially abundant phosphoproteins (DAPPs) were associated with microtubule cytoskeleton organization and spermatid differentiation. The DAPPs included proteins already known to be associated with spermatogenesis, as well as many not previously studied during this process. Six genes coding for DAPPs were knocked down, respectively, in Wolbachia-free fly testes. Among them, Slmap knockdown caused the most severe damage in spermatogenesis, with no mature sperm observed in seminal vesicles. Immunofluorescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted. These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction. By identifying phospho-modulated proteins we also provide a significant candidate set for future studies on their roles in spermatogenesis.
Ilyin, A. A., Kononkova, A. D., Golova, A. V., Shloma, V. V., Olenkina, O. M., Nenasheva, V. V., Abramov, Y. A., Kotov, A. A., Maksimov, D. A., Laktionov, P. P., Pindyurin, A. V., Galitsyna, A. A., Ulianov, S. V., Khrameeva, E. E., Gelfand, M. S., Belyakin, S. N., Razin, S. V. and Shevelyov, Y. Y. (2022). Comparison of genome architecture at two stages of male germline cell differentiation in Drosophila. Nucleic Acids Res 50(6): 3203-3225. PubMed ID: 35166842
Summary:
Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). This study identified LADs and TADs at two stages of Drosophila spermatogenesis - in bamΔ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). This study found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. It was also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, the specific X chromosome organization was uncovered in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.
Kato, Y., Sawada, A., Tonai, K., Tatsuno, H., Uenoyama, T. and Itoh, M. (2022). A new allele of engrailed, en(NK14), causes supernumerary spermathecae in Drosophila melanogaster. Genes Genet Syst. PubMed ID: 35264511
Summary:
A spontaneous mutation, enNK14, was a new allele of engrailed en in Drosophila melanogaster. Females of enNK14 have three spermathecae, instead of two in wild type, under a wide range of developmental temperatures, while the males show no abnormal phenotype. Spermathecae of the mutant female can accept inseminated sperms, albeit with a delay of at least an hour until full acceptance compared with wild type. The time course of decrease in the number of stored sperms was thoroughly similar between the mutant and wild type. enNK14 females produced fewer progeny than wild type females despite storing a larger number of sperms. The delay of sperm entry and lower fecundity suggested some functional defects in secretory products of the spermathecae. In addition, some spermathecae in the mutant were accompanied by a mass of brown pigments in the adipose tissue surrounding the capsule. Six contiguous amino acids, Ser340-Ala345, were replaced by one Thr in enNK14. In another mutant, en(spt), Ser325 was also shown to be substituted by a Cys. These amino acid changes were located within a serine-rich region, in which Ser325, Ser340 and Thr341 were suggested as targets of Protein Kinase C by an in silico analysis. The splicing pattern of en mRNA did not differ between enNK14 and wild type in embryo, larva, pupa or adult. These results suggest that en plays an important role in determining the number of spermathecae as well as in sperm storage function in the Drosophila female.
McCullough, E. L., Whittington, E., Singh, A., Pitnick, S., Wolfner, M. F. and Dorus, S. (2022). The life history of Drosophila sperm involves molecular continuity between male and female reproductive tracts. Proc Natl Acad Sci U S A 119(11): e2119899119. PubMed ID: 35254899
Summary:
In species with internal fertilization, sperm spend an important part of their lives within the female. To examine the life history of the sperm during this time, semiquantitative proteomics and sex-specific isotopic labeling were used in fruit flies to determine the extent of molecular continuity between male and female reproductive tracts and provide a global catalog of sperm-associated proteins. Multiple seminal fluid proteins and female proteins associate with sperm immediately after mating. Few seminal fluid proteins remain after long-term sperm storage, whereas female-derived proteins constitute one-fifth of the postmating sperm proteome by then. These data reveal a molecular "hand-off" from males to females, which is postulated to be an important component of sperm-female interactions.
Shore, T., Levi, T., ..., Deshpande, G. and Gerlitz, O. (2022). Nucleoporin107 mediates female sexual differentiation via Dsx. Elife 11. PubMed ID: 35311642
Summary:
A missense mutation in Nucleoporin107 (Nup107; D447N) underlies XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells.

Thursday, June 2nd - Disease models

Loganathan, S., Wilson, B. A., Carey, S. B., Manzo, E., Joardar, A., Ugur, B. and Zarnescu, D. C. (2022). TDP-43 Proteinopathy Causes Broad Metabolic Alterations including TCA Cycle Intermediates and Dopamine Levels in Drosophila Models of ALS. Metabolites 12(2). PubMed ID: 35208176
Summary:
Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. This study used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, global metabolomic profiling was performed in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. This study reports alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. It was also shown that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43(G298S), and it was found that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.
Phillips, M. A., Arnold, K. R., Vue, Z., Beasley, H. K., Garza-Lopez, E., Marshall, A. G., Morton, D. J., McReynolds, M. R., Barter, T. T. and Hinton, A., Jr. (2022). Combining Metabolomics and Experimental Evolution Reveals Key Mechanisms Underlying Longevity Differences in Laboratory Evolved Drosophila melanogaster Populations. Int J Mol Sci 23(3). PubMed ID: 35162994
Summary:
Experimental evolution with Drosophila melanogaster has been used extensively for decades to study aging and longevity. In recent years, the addition of DNA and RNA sequencing to this framework has allowed researchers to leverage the statistical power inherent to experimental evolution to study the genetic basis of longevity itself. Here, we incorporated metabolomic data into to this framework to generate even deeper insights into the physiological and genetic mechanisms underlying longevity differences in three groups of experimentally evolved D. melanogaster populations with different aging and longevity patterns. Metabolomic analysis found that aging alters mitochondrial metabolism through increased consumption of NAD(+) and increased usage of the TCA cycle. Combining the genomic and metabolomic data produced a list of biologically relevant candidate genes. Among these candidates, significant enrichment was found for genes and pathways associated with neurological development and function, and carbohydrate metabolism. While enrichment for aging canonical genes was not specifically found, neurological dysregulation and carbohydrate metabolism are both known to be associated with accelerated aging and reduced longevity. Taken together, these results provide plausible genetic mechanisms for what might be driving longevity differences in this experimental system. More broadly, these findings demonstrate the value of combining multiple types of omic data with experimental evolution when attempting to dissect mechanisms underlying complex and highly polygenic traits such as aging.
Poetini, M. R., Musachio, E. A. S., Araujo, S. M., Bortolotto, V. C., Meichtry, L. B., Silva, N. C., Janner, D. E., La Rosa Novo, D., Mesko, M. F., Roehrs, R., Ramborger, B. P. and Prigol, M. (2022). Improvement of non-motor and motor behavioral alterations associated with Parkinson-like disease in Drosophila melanogaster: Comparative effects of treatments with hesperidin and L-dopa. Neurotoxicology 89: 174-183. PubMed ID: 35167856
Summary:
Non-motor alterations such as anxiety and memory deficit may represent early indications of Parkinson's disease (PD), and therapeutic strategies that reduce non-motor alterations are promising alternatives for the treatment. Therefore, the search for natural compounds that act on motor and non-motor complications is highly relevant. In this sense, this study has demonstrated the role of hesperidin (Hsd) as a citrus flavonoid and its pharmacological properties as an antioxidant and neuroprotective agent. The objective was to evaluate Hsd in developing motor and non-motor alterations in a Drosophila melanogaster model of Parkinson-like disease induced by iron (Fe) exposure. The flies were divided into six groups: control, Hsd (10 μM), L-dopa (positive control, 1 mM), Fe (1 mM), Fe + Hsd, and Fe + L-dopa. Motor coordination tests, memory assessment through aversive phototaxy, and anxiety-like behaviors characterized in flies, such as grooming and aggressiveness, were performed. The Hsd attenuated motor and non-motor alterations, such as motor coordination, memory deficits and anxiety-like behaviors, attenuated monoaminergic deficits, and lowered Fe levels in the head of flies. In addition, Hsd prolonged the life of the flies, thereby standing out from the L-dopa-treated group. Thus, Hsd can protect the dopaminergic system from insults caused by Fe, preventing non-motor alterations in PD; Hsd also reduced Fe levels in the flies' heads, suggesting that iron chelation may represent an important mechanism of action, in addition to its antioxidant action.
Lu, S., Hernan, R., Marcogliese, P. C., Huang, Y., Gertler, T. S., Akcaboy, M., Liu, S., Chung, H. L., Pan, X., Sun, X., Oguz, M. M., Oztoprak, U., de Baaij, J. H. F., Ivanisevic, J., McGinnis, E., Guillen Sacoto, M. J., Chung, W. K. and Bellen, H. J. (2022). Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures. Am J Hum Genet 109(4): 571-586. PubMed ID: 35240055
Summary:
TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. This study describes five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. The Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. The function associated with three TIAM1 variants carried by two of the probands were assessed and they were compared to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, this study provides evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.
Marcogliese, P. C., Deal, S. L., Andrews, J., ..., Marom, R., Wangler, M. F. and Yamamoto, S. (2022). Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases. Cell Rep 38(11): 110517. PubMed ID: 35294868
Summary:
Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear.This study functionally tested the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. 79 ASD variants were examined in 74 genes identified in the Simons Simplex Collection and found 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.
Overman, K. E., Choi, D. M., Leung, K., Shaevitz, J. W. and Berman, G. J. (2022). Measuring the repertoire of age-related behavioral changes in Drosophila melanogaster. PLoS Comput Biol 18(2): e1009867. PubMed ID: 35202388
Summary:
Aging affects almost all aspects of an organism-its morphology, its physiology, its behavior. Isolating which biological mechanisms are regulating these changes, however, has proven difficult, potentially due to our inability to characterize the full repertoire of an animal's behavior across the lifespan. Using data from fruit flies (D. melanogaster) this study measured the full repertoire of behaviors as a function of age. A sexually dimorphic pattern of changes was observed in the behavioral repertoire during aging. Although the stereotypy of the behaviors and the complexity of the repertoire overall remains relatively unchanged, evidence was found that the observed alterations in behavior can be explained by changing the fly's overall energy budget, suggesting potential connections between metabolism, aging, and behavior.

Wednesday June 1st - Behavior

Lin, H. H., Kuang, M. C., Hossain, I., Xuan, Y., Beebe, L., Shepherd, A. K., Rolandi, M. and Wang, J. W. (2022). A nutrient-specific gut hormone arbitrates between courtship and feeding. Nature 602(7898): 632-638. PubMed ID: 35140404
Summary:
Animals must set behavioural priority in a context-dependent manner and switch from one behaviour to another at the appropriate moment. This study probed the molecular and neuronal mechanisms that orchestrate the transition from feeding to courtship in Drosophila melanogaster. It was found that feeding is prioritized over courtship in starved males, and the consumption of protein-rich food rapidly reverses this order within a few minutes. At the molecular level, a gut-derived, nutrient-specific neuropeptide hormone-Diuretic hormone 31 (Dh31)-propels a switch from feeding to courtship. The underlying kinetics were addressed with calcium imaging experiments. Amino acids from food acutely activate Dh31(+) enteroendocrine cells in the gut, increasing Dh31 levels in the circulation. In addition, three-photon functional imaging of intact flies shows that optogenetic stimulation of Dh31(+) enter oendocrine cells rapidly excites a subset of brain neurons that express Dh31 receptor. Gut-derived Dh31 excites the brain neurons through the circulatory system within a few minutes, in line with the speed of the feeding-courtship behavioural switch. At the circuit level, there are two distinct populations of Dh31R(+) neurons in the brain, with one population inhibiting feeding through allatostatin-C and the other promoting courtship through corazonin. Together, these findings illustrate a mechanism by which the consumption of protein-rich food triggers the release of a gut hormone, which in turn prioritizes courtship over feeding through two parallel pathways.
Ning, J., Li, Z., Zhang, X., Wang, J., Chen, D., Liu, Q. and Sun, Y. (2022). Behavioral signatures of structured feature detection during courtship in Drosophila. Curr Biol 32(6): 1211-1231.e1217. PubMed ID: 35139360
Summary:
Many animals detect other individuals effortlessly. In Drosophila, previous studies have examined sensory processing during social interactions using simple blobs as visual stimulation; however, whether and how flies extract higher-order features from conspecifics to guide behavior remains elusive. Arguing that this should be reflected in sensorimotor relations, this study developed unbiased machine learning tools for natural behavior quantification and applied these tools, which may prove broadly useful, to study interacting pairs. By transforming motor patterns with female-centered reference frames, this study established circling, where heading and traveling directions intersect, as a unique pattern of social interaction during courtship. Circling was found to be highly visual, with males exhibiting view-tuned motor patterns. Interestingly, males select specific wing and leg actions based on the positions and motions of the females' heads and tails. Using system identification, visuomotor transformation functions were derived indicating history-dependent action selection, with distance predicting action initiation and angular position predicting wing choices and locomotion directions. Integration of vision with somatosensation further boosts these sensorimotor relations. Essentially comprised of orchestrated wing and leg maneuvers that are more variable in the light, circling induces mutually synchronized conspecific responses stronger than wing extension alone. Finally, this study found that actions depend on integrating spatiotemporally structured features with goals. Altogether, we identified a series of sensorimotor relations during circling, implying that during courtship, flies detect complex spatiotemporally structured features of conspecifics, laying the foundation for a mechanistic understanding of conspecific recognition in Drosophila.
Negelspach, D. C., Kaladchibachi, S., Dollish, H. K. and Fernandez, F. X. (2022). Sleep Deprivation Does Not Influence Photic Resetting of Circadian Activity Rhythms in Drosophila Clocks. Sleep 4(1): 202-207. PubMed ID: 35323172
Summary:
Previous investigations in humans and rodent animal models have assessed the interplay of sleep in the circadian system's phase responses to nighttime light exposure. The resulting data have been mixed, but generally support a modulatory role for sleep in circadian photic resetting (not an absolute requirement). Drosophila have been historically used to provide important insights in the sleep and circadian sciences. However, no experiments to date have evaluated how immediate sleep need or recent sleep history affects their pacemaker's phase readjustments to light. We did so in the current study by (1) forcing separate groups of animals to stay awake for 1 or 4 h after they were shown a broadspectrum pulse (15 min during the first half of the night, 950 lux), or (2) placing them on a restricted sleep schedule for a week before light presentation without any subsequent sleep disruption. Forced sleep restriction, whether acute or chronic, did not alter the size of light-induced phase shifts. These data are consistent with observations made in other diurnal animals and raise the possibility, more broadly, that phototherapies applied during sleep-such as may be necessary during the winter months-may still be efficacious in individuals experiencing sleep-continuity problems such as insomnia.
Pan, Y., Li, W., Deng, Z., Sun, Y., Ma, X., Liang, R., Guo, X., Sun, Y., Li, W., Jiao, R. and Xue, L. (2022). Myc suppresses male-male courtship in Drosophila. Embo j 41(7): e109905. PubMed ID: 35167135
Summary:
Despite strong natural selection on species, same-sex sexual attraction is widespread across animals, yet the underlying mechanisms remain elusive. This study reports that the proto-oncogene Myc is required in dopaminergic neurons to inhibit Drosophila male-male courtship. Loss of Myc, either by mutation or neuro-specific knockdown, induced males' courtship propensity toward other males. Genetic screen identified DOPA decarboxylase (Ddc) as a downstream target of Myc. While loss of Ddc abrogated Myc depletion-induced male-male courtship, Ddc overexpression sufficed to trigger such behavior. Furthermore, Myc-depleted males exhibited elevated dopamine level in a Ddc-dependent manner, and their male-male courtship was blocked by depleting the dopamine receptor DopR1. Moreover, Myc directly inhibits Ddc transcription by binding to a target site in the Ddc promoter, and deletion of this site by genome editing was sufficient to trigger male-male courtship. Finally, drug-mediated Myc depletion in adult neurons by GeneSwitch technique sufficed to elicit male-male courtship. Thus, this study uncovered a novel function of Myc in preventing Drosophila male-male courtship, and supports the crucial roles of genetic factors in inter-male sexual behavior.
Kemppainen, J., Scales, B., Razban Haghighi, K., Takalo, J., Mansour, N., McManus, J., Leko, G., Saari, P., Hurcomb, J., Antohi, A., Suuronen, J. P., Blanchard, F., Hardie, R. C., Song, Z., Hampton, M., Eckermann, M., Westermeier, F., Frohn, J., Hoekstra, H., Lee, C. H., Huttula, M., Mokso, R. and Juusola, M. (2022). Binocular mirror-symmetric microsaccadic sampling enables Drosophila hyperacute 3D vision. Proc Natl Acad Sci U S A 119(12): e2109717119. PubMed ID: 35298337
Summary:
To move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies' depth-perception dynamics, limits, and visual behaviors.
Kemppainen, J., Mansour, N., Takalo, J. and Juusola, M. (2022). High-speed imaging of light-induced photoreceptor microsaccades in compound eyes. Commun Biol 5(1): 203. PubMed ID: 35241794
Summary:
ide compound eyes, photoreceptors contract to light changes, sharpening retinal images of the moving world in time. Current methods to measure these so-called photoreceptor microsaccades in living insects are spatially limited and technically challenging. this study present goniometric high-speed deep pseudopupil (GHS-DPP) microscopy to assess how the rhabdomeric insect photoreceptors and their microsaccades are organised across the compound eyes. This method enables non-invasive rhabdomere orientation mapping, whilst their microsaccades can be locally light-activated, revealing the eyes' underlying active sampling motifs. By comparing the microsaccades in wild-type Drosophila's open rhabdom eyes to spam-mutant eyes, reverted to an ancestral fused rhabdom state, and honeybee's fused rhabdom eyes, this study showed how different eye types sample light information. These results show different ways compound eyes initiate the conversion of spatial light patterns in the environment into temporal neural signals and highlight how this active sampling can evolve with insects' visual needs.

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