What's hot today: Current papers in developmental biology and gene function

What's hot today:
Current papers in developmental biology and gene function

Friday, January 29th, 2021 - Disease models

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Savitsky, M., Solis, G. P., Kryuchkov, M. and Katanaev, V. L. (2020). Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies. Biomedicines 8(10). PubMed ID: 33036271
Summary:
Several hundred genes have been identified to contribute to epilepsy-the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, this study describes the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the Drosophila functions. This genetic model will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics.

Aragao Gomes, L., Uytterhoeven, V., ...., Verstreken, P. and Thal, D. R. (2021). Maturation of neuronal AD-tau pathology involves site-specific phosphorylation of cytoplasmic and synaptic tau preceding conformational change and fibril formation. Acta Neuropathol. PubMed ID: 33427938
Summary:
It is not yet clear which events initiate the early preclinical phase of Alzheimer's disease (AD) tauopathy and whether they have impact on the propagation of tau (see Drosophila Tau) pathology in later disease stages. To address this question, the distribution was analyzed of tau species phosphorylated at T231, S396/S404 and S202/T205, conformationally modified at the MC1 epitope, and fibrillary tau was detected by the Gallyas method (Gallyas-tau), in the brains of 15 symptomatic and 20 asymptomatic cases with AD pathology as well as of 19 nonAD cases. As initial tau lesions, phosphorylated-T231-tau was identified diffusely distributed within the somatodendritic compartment (IC-tau) and phosphorylated-S396/pS404-tau in axonal lesions of the white matter and in the neuropil (IN-tau). The subcellular localization of pT231-tau in the cell body and pS396/pS404-tau in the presynapse was confirmed in hP301L mutant Drosophila larvae. Phosphorylated-S202/T205-tau, MC1-tau and Gallyas-tau were negative for these lesions. IC- and IN-tau were observed in all analyzed regions of the human brain, including early affected regions in nonAD cases (entorhinal cortex) and late affected regions in symptomatic AD cases (cerebellum), indicating that tau pathology initiation follows similar processes when propagating into previously unaffected regions. Furthermore, a sequence of AD-related maturation of tau-aggregates was observed, initiated by the appearance of IC- and IN-tau, followed by the formation of pretangles exhibiting pT231-tau, pS396/pS404-tau and pS202/pT205-tau, then by MC1-conformational tau, and, finally, by the formation of Gallyas-positive NFTs. Since cases classified as nonAD already showed IC- and IN-tau, these findings suggest that these lesions are a prerequisite for the development of AD.

Shaposhnikov, M. V., Zemskaya, N. V., Koval, L., Minnikhanova, N. R., Kechko, O. I., Mitkevich, V. A., Makarov, A. A. and Moskalev, A. (2020). Amyloid-beta peptides slightly affect lifespan or antimicrobial peptide gene expression in Drosophila melanogaster. BMC Genet 21(Suppl 1): 65. PubMed ID: 33092519
Summary:
Beta-amyloid peptide (Aβ) is the key protein in the pathogenesis of Alzheimer's disease, the most common age-related neurodegenerative disorder in humans. Aβ peptide induced pathological phenotypes in different model organisms include neurodegeneration and lifespan decrease. However, recent experimental evidence suggests that Aβ may utilize oligomerization and fibrillization to function as an antimicrobial peptide (AMP), and protect the host from infections. This study used the power of Drosophila model to study mechanisms underlying a dual role for Aβ peptides. The effects were investigated of Drosophila treatment with three Aβ42 peptide isoforms, which differ in their ability to form oligomers and aggregates on the lifespan, locomotor activity and AMP genes expression. Aβ42 slightly decreased female's median lifespan (by 4.5%), but the effect was not related to the toxicity of peptide isoform. The lifespan and relative levels of AMP gene expression in male flies as well as locomotor activity in both sexes were largely unaffected by Aβ42 peptide treatment. Regardless of the effects on lifespan, Aβ42 peptide treatment induced decrease in AMP genes expression in females, but the effects were not robust. The results demonstrate that chronic treatment with Aβ42 peptides does not drastically affect fly aging or immunity.

Bawa, S., Gameros, S., Baumann, K., Brooks, D. S., Kollhoff, J. A., Zolkiewski, M., David Re Cecconi, A., Panini, N., Russo, M., Piccirillo, R., Johnson, D. K., Kashipathy, M. M., Battaile, K. P., Lovell, S., Bouyain, S. E. A., Kawakami, J. and Geisbrecht, E. R. (2020). Costameric Integrin and Sarcoglycan protein levels are altered in a Drosophila model for Limb Girdle Muscular Dystrophy type 2H. Mol Biol Cell: mbcE20070453. PubMed ID: 33296226
Summary:
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. Previous work established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. This study shows that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N and 520fs) induce myofibril abnormalities, altered nuclear morphology and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, the protein levels of βPS integrin and Sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant, aberrantly accumulate α- and β-Dystroglycan and α-Sarcoglycan. It is speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Zhao, J., Petitjean, D., Haddad, G. A., Batulan, Z. and Blunck, R. (2020). A Common Kinetic Property of Mutations Linked to Episodic Ataxia Type 1 Studied in the Shaker Kv Channel. Int J Mol Sci 21(20). PubMed ID: 33066705
Summary:
Episodic ataxia type 1 is caused by mutations in the KCNA1 gene encoding for the voltage-gated potassium channel Kv1.1. There have been many mutations in Kv1.1 linked to episodic ataxia reported and typically investigated by themselves or in small groups. The aim of this article is to determine whether it is possible to define a functional parameter common to all Kv1.1 mutants that have been linked to episodic ataxia. The disease mutations linked to episodic ataxia were introduced in the drosophila analog of Kv1.1, the Shaker Kv channel, and the channels were expressed in Xenopus oocytes. Using the cut-open oocyte technique, the gating and ionic currents were characterized. The episodic ataxia mutations were found to variably alter the different gating mechanisms described for Kv channels. The common characteristic was a conductance voltage relationship and inactivation shifted to less polarized potentials. It is suggested that a combination of a prolonged action potential and slowed and incomplete inactivation leads to development of ataxia when Kv channels cannot follow or adapt to high firing rates.

Park, J. E., Tron, T. X. T., Park, N., Yeom, J., Kim, K. and Kang, M. J. (2020). The Function of Drosophila USP14 in Endoplasmic Reticulum Stress and Retinal Degeneration in a Model for Autosomal Dominant Retinitis Pigmentosa. Biology (Basel) 9(10). PubMed ID: 33053617
Summary:
Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. This study analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, it was observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress.

Thursday, January 28th - Signaling

Aragao Gomes, L., Uytterhoeven, V., Lopez-Sanmartin, D., Tome, S. O., Tousseyn, T., Vandenberghe, R., Vandenbulcke, M., von Arnim, C. A. F., Verstreken, P. and Thal, D. R. (2021). Maturation of neuronal AD-tau pathology involves site-specific phosphorylation of cytoplasmic and synaptic tau preceding conformational change and fibril formation. Acta Neuropathol. PubMed ID: 33427938
Summary:
In Alzheimer's disease (AD), tau-protein (see Drosophila Tau) undergoes a multi-step process involving the transition from a natively unfolded monomer to large, aggregated structures such as neurofibrillary tangles (NFTs). However, it is not yet clear which events initiate the early preclinical phase of AD tauopathy and whether they have impact on the propagation of tau pathology in later disease stages. To address this question, the distribution was analyzed of tau species phosphorylated at T231, S396/S404 and S202/T205, conformationally modified at the MC1 epitope, and fibrillary tau was detected by the Gallyas method (Gallyas-tau), in the brains of 15 symptomatic and 20 asymptomatic cases with AD pathology as well as of 19 nonAD cases. As initial tau lesions, phosphorylated-T231-tau was identified diffusely distributed within the somatodendritic compartment (IC-tau) and phosphorylated-S396/pS404-tau in axonal lesions of the white matter and in the neuropil (IN-tau). The subcellular localization of pT231-tau in the cell body and pS396/pS404-tau in the presynapse was confirmed in hP301L mutant Drosophila larvae. Phosphorylated-S202/T205-tau, MC1-tau and Gallyas-tau were negative for these lesions. IC- and IN-tau were observed in all analyzed regions of the human brain, including early affected regions in nonAD cases (entorhinal cortex) and late affected regions in symptomatic AD cases (cerebellum), indicating that tau pathology initiation follows similar processes when propagating into previously unaffected regions. Furthermore, a sequence of AD-related maturation of tau-aggregates was observed, initiated by the appearance of IC- and IN-tau, followed by the formation of pretangles exhibiting pT231-tau, pS396/pS404-tau and pS202/pT205-tau, then by MC1-conformational tau, and, finally, by the formation of Gallyas-positive NFTs. Since cases classified as nonAD already showed IC- and IN-tau, these findings suggest that these lesions are a prerequisite for the development of AD.

Thursday, January 28th - Signaling

Chen, W. T., Yang, H. Y., Lin, C. Y., Lee, Y. Z., Ma, S. C., Chen, W. C. and Yin, H. S. (2020). Structural Insight into the Contributions of the N-Terminus and Key Active-Site Residues to the Catalytic Efficiency of Glutamine Synthetase 2. Biomolecules 10(12). PubMed ID: 33327463
Summary:
Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. This study reports the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3-5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. This study mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.

Blenau, W., Wilms, J. A., Balfanz, S. and Baumann, A. (2020). AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity. Int J Mol Sci 21(24). PubMed ID: 33302363
Summary:
The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or β-adrenergic-like octopamine receptors. Currently, one α- and four β-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α(2)-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctalpha2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP](i)). This study shows that the orthologous receptor of the honeybee (AmOct&alpha2R), phylogenetically groups in a clade closely related to human α(2)-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP](i). The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.

Bellec, K., Pinot, M., Gicquel, I. and Le Borgne, R. (2021). The Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila sensory organ precursors cells. Development 148(1). PubMed ID: 33298463
Summary:
Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. Previously work has shown that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. This study reports that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, increased amounts of signalling-competent Notch as well as Delta and Sanpodo were observed at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. It is proposes that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.

Cui, L., Song, W., Zeng, Y., Wu, Q., Fan, Z., Huang, T., Zeng, B., Zhang, M., Ni, Q., Li, Y., Wang, T., Li, D., Mao, X., Lian, T., Yang, D., Yang, M. and Fan, X. (2020). Deubiquitinase USP7 regulates Drosophila aging through ubiquitination and autophagy. Aging (Albany NY) 12(22): 23082-23095. PubMed ID: 33221768
Summary:
Ubiquitination-mediated protein degradation is the selective degradation of diverse forms of damaged proteins that are tagged with ubiquitin, while deubiquitinating enzymes reverse ubiquitination-mediated protein degradation by removing the ubiquitin chain from the target protein. The interactions of ubiquitinating and deubiquitinating enzymes are required to maintain protein homeostasis. The ubiquitin-specific protease USP7 is a deubiquitinating enzyme that indirectly plays a role in repairing DNA damage and development. However, the mechanism of its participation in aging has not been fully explored. Regarding this issue, this study found that USP7 was necessary to maintain the normal lifespan of Drosophila melanogaster, and knockdown of dusp7 shortened the lifespan and reduced the ability of Drosophila to cope with starvation, oxidative stress and heat stress. Furthermore, this study showed that the ability of USP7 to regulate aging depends on the autophagy and ubiquitin signaling pathways. Furthermore, 2,5-dimethyl-celecoxib (DMC), a derivative of celecoxib, can partially restore the shortened lifespan and aberrant phenotypes caused by dusp7 knockdown. These results suggest that USP7 is an important factor involved in the regulation of aging, and related components in this regulatory pathway may become new targets for anti-aging treatments.

Besen-McNally, R., Gjelsvik, K. J. and Losick, V. P. (2020). Wound-induced polyploidization is dependent on integrin-yki signaling. Biol Open. PubMed ID: 33355119
Summary:
A key step in tissue repair is to replace lost or damaged cells. This occurs via two strategies: restoring cell number through proliferation or increasing cell size through polyploidization. Studies in Drosophila and vertebrates have demonstrated that polyploid cells arise in adult tissues, at least in part, to promote tissue repair and restore tissue mass. However, the signals that cause polyploid cells to form in response to injury remain poorly understood. In the adult Drosophila epithelium, wound-induced polyploid cells are generated by both cell fusion and endoreplication, resulting in a giant polyploid syncytium. This study identified the integrin focal adhesion complex as an activator of wound-induced polyploidization. Both integrin and focal adhesion kinase are upregulated in the wound-induced polyploid cells and are required for Yorkie induced endoreplication and cell fusion. As a result, wound healing is perturbed when focal adhesion genes are knocked down. These findings show that conserved focal adhesion signaling is required to initiate wound-induced polyploid cell growth.

Chen, W., Shen, Z., Asteriti, S., Chen, Z., Ye, F., Sun, Z., Wan, J., Montell, C., Hardie, R. C., Liu, W. and Zhang, M. (2020). Calmodulin binds to Drosophila TRP with an unexpected mode. Structure. PubMed ID: 33326749
Summary:
Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca(2+) mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca(2+) modulated CaM/TRP interaction is poorly understood. This study discovered an unexpected, Ca(2+)-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca(2+)-mediated feedback on the channel activity. Finally, TRPC4, the closest mammalian paralog of Drosophila TRP, was found to adopt a similar CaM binding mode.

Wednesday, December 27th - Adult physiology

Simard, C. J., Touaibia, M., Allain, E. P., Hebert-Chatelain, E. and Pichaud, N. (2020). Role of the Mitochondrial Pyruvate Carrier in the Occurrence of Metabolic Inflexibility in Drosophila melanogaster Exposed to Dietary Sucrose. Metabolites 10(10). PubMed ID: 33066485
Summary:
Excess dietary carbohydrates are linked to dysregulation of metabolic pathways converging to mitochondria and metabolic inflexibility. This study determined the role of the mitochondrial pyruvate carrier (MPC) in the occurrence of this metabolic inflexibility in wild-type (WT) and MPC1-deficient (MPC1(def)) flies that were exposed to diets with different sucrose concentrations for 15-25 days (Standard Diet: SD, Medium-Sucrose Diet: MSD, and High-Sucrose Diet: HSD). The results showed that MPC1(def) flies had lower mitochondrial respiration rates than WT flies on the SD and MSD. However, when exposed to the HSD, WT flies displayed decreased mitochondrial respiration rates compared to MPC1(def) flies. WT flies exposed to the HSD also displayed increased proline contribution and slightly decreased MPC1 expression. Surprisingly, when fed the MSD and the HSD, few metabolites were altered in WT flies whereas MPC1(def) flies display significant accumulation of glycogen, glucose, fructose, lactate, and glycerol. Overall, this suggests that metabolic inflexibility starts to occur in WT flies after 15-25 days of exposure to the HSD whereas the MPC1(def) flies display metabolic inflexibility independently of the diet provided. This study thus highlights the involvement of MPC as an essential protein in Drosophila to maintain proper metabolic homeostasis during changes in dietary resources.

Chen, F., Su, R., Ni, S., Liu, Y., Huang, J., Li, G., Wang, Q., Zhang, X. and Yang, Y. (2021). Context-dependent responses of Drosophila intestinal stem cells to intracellular reactive oxygen species. Redox Biol 39: 101835. PubMed ID: 33360688
Summary:
Stem cells have unique Reactive oxygen species (ROS) regulation while cancer cells frequently show a constitutive oxidative stress that is associated with the invasive phenotype. Antioxidants have been proposed to forestall tumor progression while targeted oxidants have been used to destroy tumor cells. This study used Drosophila midgut intestinal stem cell (ISCs) to tackle this question. The ROS levels of ISCs remained low in comparison to that of differentiated cells and increased with ageing, which was accompanied by elevated proliferation of ISCs in aged Drosophila. Neither upregulation nor downregulation of ROS levels significantly affected ISCs, implicating an intrinsic homeostatic range of ROS in ISCs. Interestingly, similar moderately elevated ROS levels were observed in both tumor-like ISCs induced by Notch (N) depletion and extracellular matrix (ECM)-deprived ISCs induced by β-integrin (mys) depletion. Elevated ROS levels further promoted the proliferation of tumor-like ISCs while reduced ROS levels suppressed the hyperproliferation phenotype; on the other hand, further increased ROS facilitated the survival of ECM-deprived ISCs while reduced ROS exacerbated the loss of ECM-deprived ISCs. However, N- and mys-depleted ISCs, which resembled metastatic tumor cells, harbored even higher ROS levels and were subjected to more severe cell loss, which could be partially prevented by ectopic supply of antioxidant enzymes, implicating a delicate pro-surviving and proliferating range of ROS levels for ISCs. Taken together, these results revealed stem cells can differentially respond to distinct ROS levels.

Zhao, H. and Wang, T. (2020). PE homeostasis rebalanced through mitochondria-ER lipid exchange prevents retinal degeneration in Drosophila. PLoS Genet 16(10): e1009070. PubMed ID: 33064773
Summary:
The major glycerophospholipid phosphatidylethanolamine (PE) in the nervous system is essential for neural development and function. There are two major PE synthesis pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum (ER) and the phosphatidylserine decarboxylase (PSD) pathway in mitochondria. However, the role played by mitochondrial PE synthesis in maintaining cellular PE homeostasis is unknown. This study shows that Drosophila pect (phosphoethanolamine cytidylyltransferase) mutants lacking the CDP-ethanolamine pathway, exhibited alterations in phospholipid composition, defective phototransduction, and retinal degeneration. Induction of the PSD pathway fully restored levels and composition of cellular PE, thus rescued the retinal degeneration and defective visual responses in pect mutants. Disrupting lipid exchange between mitochondria and ER blocked the ability of PSD to rescue pect mutant phenotypes. These findings provide direct evidence that the synthesis of PE in mitochondria contributes to cellular PE homeostasis, and suggest the induction of mitochondrial PE synthesis as a promising therapeutic approach for disorders associated with PE deficiency.

Yamada, T., Hironaka, K. I., Habara, O., Morishita, Y. and Nishimura, T. (2020). A developmental checkpoint directs metabolic remodelling as a strategy against starvation in Drosophila. Nat Metab 2(10): 1096-1112. PubMed ID: 33046910
Summary:
Steroid hormones are crucial regulators of life-stage transitions during development in animals. However, the molecular mechanisms by which developmental transition through these stages is coupled with optimal metabolic homeostasis remains poorly understood. This study demonstrates through mathematical modelling and experimental validation that ecdysteroid-induced metabolic remodelling from resource consumption to conservation can be a successful life-history strategy to maximize fitness in Drosophila larvae in a fluctuating environment. Specifically, the ecdysteroid-inducible protein ImpL2 protects against hydrolysis of circulating trehalose following pupal commitment in larvae. Stored glycogen and triglycerides in the fat body are also conserved, even under fasting conditions. Moreover, pupal commitment dictates reduced energy expenditure upon starvation to maintain available resources, thus negotiating trade-offs in resource allocation at the physiological and behavioural levels. The optimal stage-specific metabolic shift elucidated by these predictive and empirical approaches reveals that Drosophila has developed a highly controlled system for ensuring robust development that may be conserved among higher-order organisms in response to intrinsic and extrinsic cues.

Zhao, X., Li, Y., Zhao, Z. and Du, J. (2020). Extra sex combs buffers sleep-related stresses through regulating Heat shock proteins. Faseb j. PubMed ID: 33220007
Summary:
The impact of global warming on the life of the earth is increasingly concerned. Previous studies indicated that temperature changes have a serious impact on insect sleep. Sleep is critical for animals as it has many important physiological functions. It is of great significance to study the regulation mechanism of temperature-induced sleep changes for understanding the impact of global warming on insects. More importantly, understanding how these pressures regulate sleep can provide insights into improving sleep. This study found that extra sex combs (ESC) is a regulatory factor in this process. The data showed that ESC was an upstream negative regulatory factor of Heat shock proteins (Hsps), and it could regulate sleep in mushroom and ellipsoid bodies of Drosophila. ESC mutation exaggerates the sleep change caused by temperature, while buffering the shortening of life caused by sleep deprivation. These phenotypes can be rescued by Hsps mutants. Therefore, it is concluded that the ESC buffers sleep-related stresses through regulating Hsps.

Chartschenko, E., Hugenroth, M., Akhtar, I., Droste, A., Kolkhof, P., Bohnert, M. and Beller, M. (2020). CG32803 is the fly homolog of LDAF1 and influences lipid storage in vivo. Insect Biochem Mol Biol: 103512. PubMed ID: 33307187
Summary:
The Seipin protein is a conserved key component in the biogenesis of lipid droplets (LDs). Recently, a cooperation between human Seipin and the Lipid droplet assembly factor 1 (LDAF1) was described. LDAF1 physically interacts with Seipin and the holocomplex safeguards regular LD biogenesis. The function of LDAF1 proteins outside mammals is less clear. In yeast, the lipid droplet organization (LDO) proteins, which also cooperate with Seipin, are the putative homologs of LDAF1. While certain functional aspects are shared between the LDO and mammalian LDAF1 proteins, the relationship between the proteins is under debate. This study identified the Drosophila melanogaster protein CG32803, which was re-named to dmLDAF1, as an insect member of this protein family. dmLDAF1 decorates LDs in cultured cells and in vivo and the protein is linked to the fly and mouse Seipin proteins. Altering the dmLDAF1 abundance affects LD size, number and overall lipid storage amounts. These results suggest that the LDAF1 proteins thus fulfill an evolutionarily conserved function in the biogenesis and biology of LDs.

Tuesday, January 26th - Embryonic Development

Casani, S., Casanova, J. and Llimargas, M. (2020). Unravelling the distinct contribution of cell shape changes and cell intercalation to tissue morphogenesis: the case of the Drosophila trachea. Open Biol 10(11): 200329. PubMed ID: 33234070
Summary:
Intercalation allows cells to exchange positions in a spatially oriented manner in an array of diverse processes, spanning convergent extension in embryonic gastrulation to the formation of tubular organs. However, given the co-occurrence of cell intercalation and changes in cell shape, it is sometimes difficult to ascertain their respective contribution to morphogenesis. A well-established model to analyse intercalation, particularly in tubular organs, is the Drosophila tracheal system. There, fibroblast growth factor (FGF) signalling at the tip of the dorsal branches generates a 'pulling' force believed to promote cell elongation and cell intercalation, which account for the final branch extension. This study used a variety of experimental conditions to study the contribution of cell elongation and cell intercalation to morphogenesis and analysed their mutual requirements. Evidence is provided that cell intercalation does not require cell elongation and vice versa. The two cell behaviours are controlled by independent but simultaneous mechanisms, and cell elongation is sufficient to account for full extension of the dorsal branch, while cell intercalation has a specific role in setting the diameter of this structure. Thus, rather than viewing changes in cell shape and cell intercalation as just redundant events that add robustness to a given morphogenetic process, this study finds that they can also act by contributing to different features of tissue architecture.

Hildebrandt, K., Bach, N., Kolb, D. and Walldorf, U. (2020). The homeodomain transcription factor Orthopedia is involved in development of the Drosophila hindgut. Hereditas 157(1): 46. PubMed ID: 33213520
Summary:
The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet. This study shows that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an Otp antibody, a detailed expression analysis during hindgut development was carried out. Otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, the mutant otp allele otp(GT) was generated by ends-out gene targeting. In addition, two EMS-induced otp alleles were isolated in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut. These data suggest that Otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn Otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells.

West, J. J. and Harris, T. J. C. (2020). The Arf-GEF Steppke promotes F-actin accumulation, cell protrusions and tissue sealing during Drosophila dorsal closure. PLoS One 15(11): e0239357. PubMed ID: 33186390
Summary:
Cytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, this study analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Inversely, Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke's previously reported regulation of myosin contractility during dorsal closure.

Strong, I. J. T., Lei, X., Chen, F., Yuan, K. and O'Farrell, P. H. (2020). Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate mid-blastula transition events at a low nuclear-cytoplasmic ratio. PLoS Biol 18(10): e3000891. PubMed ID: 33090988
Summary:
Externally deposited eggs begin development with an immense cytoplasm and a single overwhelmed nucleus. Rapid mitotic cycles restore normality as the ratio of nuclei to cytoplasm (N/C) increases. A threshold N/C has been widely proposed to activate zygotic genome transcription and onset of morphogenesis at the mid-blastula transition (MBT). To test whether a threshold N/C is required for these events, N/C increase was blocked by down-regulating cyclin/Cdk1 to arrest early cell cycles in Drosophila. Embryos that were arrested two cell cycles prior to the normal MBT activated widespread transcription of the zygotic genome including genes previously described as N/C dependent. Zygotic transcription of these genes largely retained features of their regulation in space and time. Furthermore, zygotically regulated post-MBT events such as cellularization and gastrulation movements occurred in these cell cycle-arrested embryos. These results are not compatible with models suggesting that these MBT events are directly coupled to N/C. Cyclin/Cdk1 activity normally declines in tight association with increasing N/C and is regulated by N/C. By experimentally promoting the decrease in cyclin/Cdk1, this study uncoupled MBT from N/C increase, arguing that N/C-guided down-regulation of cyclin/Cdk1 is sufficient for genome activation and MBT.

Rich, A., Fehon, R. G. and Glotzer, M. (2020). Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos. Elife 9. PubMed ID: 33200987
Summary:
Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, this study shows that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. The results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.

Toda, S., McKeithan, W. L., Hakkinen, T. J., Lopez, P., Klein, O. D. and Lim, W. A. (2020). Engineering synthetic morphogen systems that can program multicellular patterning. Science 370(6514): 327-331. PubMed ID: 33060357
Summary:
In metazoan tissues, cells decide their fates by sensing positional information provided by specialized morphogen proteins. To explore what features are sufficient for positional encoding, it was asked whether arbitrary molecules (e.g., green fluorescent protein or mCherry) could be converted into synthetic morphogens. Synthetic morphogens expressed from a localized source formed a gradient when trapped by surface-anchoring proteins, and they could be sensed by synthetic receptors [an anti–green fluorescent protein (GFP) synNotch receptor]. Despite their simplicity, these morphogen systems yielded patterns reminiscent of those observed in vivo. Gradients could be reshaped by altering anchor density or by providing a source of competing inhibitor. Gradient interpretation could be altered by adding feedback loops or morphogen cascades to receiver cell response circuits. Orthogonal cell-cell communication systems provide insight into morphogen evolution and a platform for engineering tissues.

Monday, January 25th - Methods

Allen, S. E., Koreman, G. T., Sarkar, A., Wang, B., Wolfner, M. F. and Han, C. (2021). Versatile CRISPR/Cas9-mediated mosaic analysis by gRNA-induced crossing-over for unmodified genomes. PLoS Biol 19(1): e3001061. PubMed ID: 33444322
Summary:
Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. This study reports mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for 1 chromosome arm, the method's application is demonstrated in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites in the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can in principle be applied in any organism that is compatible with CRISPR/Cas9.

Spierer, A. N., Yoon, D., Zhu, C. T. and Rand, D. M. (2020). FreeClimber: Automated quantification of climbing performance in Drosophila. J Exp Biol. PubMed ID: 33188065
Summary:
Negative geotaxis (climbing) performance is a useful metric for quantifying Drosophila health. Manual methods to quantify climbing performance are tedious and often biased, while many available computational methods have challenging hardware or software requirements. An alternative is presented in this paper: FreeClimber. This open source, Python-based platform subtracts a video's static background to improve detection for flies moving across heterogeneous backgrounds. FreeClimber calculates a cohort's velocity as the slope of the most linear portion of a mean-vertical position vs. time curve. It can run from a graphical user interface for optimization or a command line interface for high-throughput and automated batch processing, improving accessibility for users with different expertise. FreeClimber outputs calculated slopes, spot locations for follow up analyses (e.g. tracking), and several visualizations and plots. FreeClimber's utility was demonstrated in a longitudinal study for endurance exercise performance in Drosophila mitonuclear genotypes using six distinct mitochondrial haplotypes paired with a common w (1118) nuclear background.

Luhur, A., Mariyappa, D., Klueg, K. M., Buddika, K., Tennessen, J. M. and Zelhof, A. C. (2020). Adapting Drosophila melanogaster Cell Lines to Serum-Free Culture Conditions. G3 (Bethesda). PubMed ID: 33028628
Summary:
Successful Drosophila cell culture relies on media containing xenogenic components such as fetal bovine serum to support continuous cell proliferation. This paper report a serum-free culture condition that supports the growth and proliferation of Drosophila S2R+ and Kc167 cell lines. Importantly, the gradual adaptation of S2R+ and Kc167 cells to a media lacking serum was supported by supplementing the media with adult Drosophila soluble extract, commonly known as fly extract. The utility of these adapted cells lines is largely unchanged. The adapted cells exhibited robust proliferative capacity and a transfection efficiency that was comparable to control cells cultured in serum-containing media. Transcriptomic data indicated that the S2R+ cells cultured with fly extract retain their hemocyte-specific transcriptome profile, and there were no global changes in the transcriptional output cell signaling pathways. Metabolome studies indicate that there were very limited metabolic changes. In fact, the cells were likely experiencing less oxidative stress when cultured in the serum-free media supplemented with fly extract. Overall, the Drosophila cell culture conditions reported in this study consequently provide researchers with an alternative and physiologically relevant resource to address cell biological research questions.

Meloni, I., Sachidanandan, D., Thum, A. S., Kittel, R. J. and Murawski, C. (2020). Controlling the behaviour of Drosophila melanogaster via smartphone optogenetics. Sci Rep 10(1): 17614. PubMed ID: 33077824
Summary:
Invertebrates such as Drosophila melanogaster have proven to be a valuable model organism for studies of the nervous system. In order to control neuronal activity, optogenetics has evolved as a powerful technique enabling non-invasive stimulation using light. This requires light sources that can deliver patterns of light with high temporal and spatial precision. Currently employed light sources for stimulation of small invertebrates, however, are either limited in spatial resolution or require sophisticated and bulky equipment. This work used smartphone displays for optogenetic control of Drosophila melanogaster. An open-source smartphone app was developed that allows time-dependent display of light patterns, and this was used to activate and inhibit different neuronal populations in both larvae and adult flies. Characteristic behavioural responses were observed depending on the displayed colour and brightness and in agreement with the activation spectra and light sensitivity of the used channelrhodopsins. By displaying patterns of light, larval movement was constrained, and it was possible to guide larvae on the display. This method serves as a low-cost high-resolution testbench for optogenetic experiments using small invertebrate species and is particularly appealing to application in neuroscience teaching labs.

Bogovic, J. A., Otsuna, H., Heinrich, L., Ito, M., Jeter, J., Meissner, G., Nern, A., Colonell, J., Malkesman, O., Ito, K. and Saalfeld, S. (2020). An unbiased template of the Drosophila brain and ventral nerve cord. PLoS One 15(12): e0236495. PubMed ID: 33382698
Summary:
The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individual neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. This work created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. The central brain template was evaluated against the four most competitive, publicly available brain templates, and it was demonstrated that this template enables more accurate registration with fewer local deformations in shorter time.

Nelson, K. A., Warder, B. N., DiNardo, S. and Anllo, L. (2020). Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad.. J Vis Exp(164). PubMed ID: 33135688
Summary:
The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. This study presents a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but insight is provided on how to prevent common mistakes and how to overcome these challenges. This is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the cells in their natural gonadal environment.

Barber, A. F. and Sehgal, A. (2021). Monitoring Electrical Activity in Drosophila Circadian Output Neurons. Methods Mol Biol 2130: 221-232. PubMed ID: 33284448
Summary:
Drosophila melanogaster is a powerful model organism used to study circadian rhythms, historically for elucidating the molecular basis of the clock and, more recently, for allowing for dissection of neural circuits underlying rhythmic behavior. The fly can be used to investigate the neuronal basis of complex behaviors at single-neuron resolution. Patch clamp electrophysiology permits single-neuron recording of resting membrane potential and action potential firing in response to genetic or environmental manipulations or application of drugs and neurotransmitters. This paper describe a protocol for dissecting Drosophila brains for electrophysiology, setting up and using a patch clamp system, and analyzing firing data around the circadian day and in stimulation-response experiments to test for functional neuronal connectivity in circadian circuits.

Bosch, J. A., Birchak, G. and Perrimon, N. (2021). . Precise genome engineering in Drosophila using prime editing. Proc Natl Acad Sci U S A 118(1). PubMed ID: 33443210
Summary:
Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. This study applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, premature stop codons were precisely introduced in three classical visible marker genes, ebony, white, and forked. Furthermore, by restricting editing to germ cells, efficient germ-line transmission of a precise edit in ebony to 36% of progeny was demonstrated. The results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.

Friday January 22 - Immune Response

Kobler, J. M., Rodriguez Jimenez, F. J., Petcu, I. and Grunwald Kadow, I. C. (2020). Immune Receptor Signaling and the Mushroom Body Mediate Post-ingestion Pathogen Avoidance. Curr Biol. PubMed ID: 33007248
Summary:
In spite of the positive effects of bacteria on health, certain species are harmful, and therefore, animals must weigh nutritional benefits against negative post-ingestion consequences and adapt their behavior accordingly. This study used Drosophila to unravel how the immune system communicates with the brain, enabling avoidance of harmful foods. Using two different known fly pathogens, mildly pathogenic Erwinia carotovora (Ecc15) and highly virulent Pseudomonas entomophila (Pe), preference behavior was analyzed in naive flies and after ingestion of either of these pathogens. Although survival assays confirmed the harmful effect of pathogen ingestion, naive flies preferred the odor of either pathogen to air and also to harmless mutant bacteria, suggesting that flies are not innately repelled by these microbes. By contrast, feeding assays showed that, when given a choice between pathogenic and harmless bacteria, flies-after an initial period of indifference-shifted to a preference for the harmless strain, a behavior that lasted for several hours. Flies lacking synaptic output of the mushroom body (MB), the fly's brain center for associative memory formation, lost the ability to distinguish between pathogenic and harmless bacteria, suggesting this to be an adaptive behavior. Interestingly, this behavior relied on the immune receptors PGRP-LC and -LE and their presence in octopaminergic neurons. A model is postulated wherein pathogen ingestion triggers PGRP signaling in octopaminergic neurons, which in turn relay the information about the harmful food source directly or indirectly to the MB, where an appropriate behavioral output is generated.

Satapathy, P., Prakash, J. K., Gowda, V. C., More, S. S., K, M., Chandramohan, V. and Zameer, F. (2020). Targeting Imd pathway receptor in Drosophila melanogaster and repurposing of phyto-inhibitors: structural modulation and molecular dynamics. J Biomol Struct Dyn: 1-12. PubMed ID: 33050786
Summary:
Dysbiosis is a major cause of disease in an individual, generally initiated in the gastrointestinal tract. The gut, also known as the second brain, constitutes a major role in immune signaling. To study the immunity cascade, the Drosophila model was considered targeting the Imd pathway receptor (2F2L) located in the midgut. This receptor further initiates the immune signaling mechanism influenced by bacteria. To inhibit the Imd pathway, the crystal structure of Imd with PDB: 2F2L was considered for the screening of suitable ligand/inhibitor. In light of previous studies, repurposing of anti-diabetic ligands from the banana plant namely lupeol (LUP), stigmasterol (STI), β-sitosterol (BST) and umbelliferone (UMB) were screened. This study identifies the potential inhibitor along with the tracheal toxin (TCT), a major peptidoglycan constituent of microbes. The molecular docking and molecular dynamics simulation of complexes 2F2L-MLD, 2F2L- CAP, 2F2L-LUP, 2F2L-BST, 2F2L-STI and 2F2L-UMB elucidates the intermolecular interaction into the inhibitory property of ligands. The results of this study infer LUP and UMB as better ligands with high stability and functionality among the screened candidates. This study provides insights into the dysbiosis and its amelioration by plant-derived molecules. The identified drugs (LUP & UMB) will probably act as an inhibitor against microbial dysbiosis and other related pathogenesis (diabetes and diabetic neuropathy). Further, this study will widen avenues in fly biology research and which could be used as a therapeutic model in the rapid, reliable and reproducible screening of phytobiologics in complementary and alternative medicine for various lifestyle associated complications.

Attieh, Z., Mouawad, C., Rejasse, A., Jehanno, I., Perchat, S., Hegna, I. K., Okstad, O. A., Kallassy Awad, M., Sanchis-Borja, V. and El Chamy, L. (2020). The fliK Gene Is Required for the Resistance of Bacillus thuringiensis to Antimicrobial Peptides and Virulence in Drosophila melanogaster. Front Microbiol 11: 611220. PubMed ID: 33391240
Summary:
Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. In this study, using a random mutagenesis approach, the fliK gene, encoding an internal molecular ruler that controls flagella hook length, was identified as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter's. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, these results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs.

Wang, Y., Yang, F., Cao, X., Zou, Z., Lu, Z., Kanost, M. R. and Jiang, H. (2020). Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta. Proc Natl Acad Sci U S A 117(38): 23581-23587. PubMed ID: 32900946
Summary:
Proteolytic activation of phenoloxidase (PO) and the cytokine Spatzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. This study presents biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta. Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. This study found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR(176)*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH(112)*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpatzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, this study has elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.

Wang, J. B., Elya, C. and St Leger, R. J. (2020). Genetic variation for resistance to the specific fly pathogen Entomophthora muscae. Sci Rep 10(1): 14284. PubMed ID: 32868814
Summary:
Substantial variation was found in resistance to the fly-specific pathogen Entomophthora muscae 'Berkeley' (Entomophthoromycota), in 20 lines from the Drosophila melanogaster Genetic Reference Panel (DGRP). Resistance to E. muscae is positively (r = 0.55) correlated with resistance to the broad host range ascomycete entomopathogen Metarhizium anisopliae (Ma549), indicative of generalist (non-specific) defenses. Most of the lines showing above average resistance to Ma549 showed cross-resistance to E. muscae. However, lines that succumbed quickly to Ma549 exhibited the full range of resistance to E. muscae. This suggests fly populations differ in E. muscae-specific resistance mechanisms as well as generic defences effective against both Ma549 and E. muscae. Trade-offs were sought that could account for inter-line variation, but increases (decreases) in disease resistance to E. muscae are not consistently associated with increases (decreases) of resistance to oxidative stress, starvation stress and sleep indices. That these pathogens are dynamic agents of selection on hosts is reflected in this genetic variation for resistance in lines derived from wild populations.

Chakrabarti, S. and Visweswariah, S. S. (2020). Intramacrophage ROS Primes the Innate Immune System via JAK/STAT and Toll Activation. Cell Rep 33(6): 108368. PubMed ID: 33176146
Summary:
Tissue injury is one of the most severe environmental perturbations for a living organism. When damage occurs in adult Drosophila, there is a local response of the injured tissue and a coordinated action across different tissues to help the organism overcome the deleterious effect of an injury. This study shows a change in the transcriptome of hemocytes at the site of tissue injury, with pronounced activation of the Toll signaling pathway. Induction of the cytokine upd-3 and Toll receptor activation occur in response to injury alone, in the absence of a pathogen. Intracellular accumulation of hydrogen peroxide in hemocytes is essential for upd-3 induction and is facilitated by the diffusion of hydrogen peroxide through a channel protein Prip. Importantly, hemocyte activation and production of reactive oxygen species (ROS) at the site of a sterile injury provide protection to flies on subsequent infection, demonstrating training of the innate immune system.

Thursday, January 22nd - Signaling

Swanson, L. C., Trujillo, E. A., Thiede, G. H., Katzenberger, R. J., Shishkova, E., Coon, J. J., Ganetzky, B. and Wassarman, D. A. (2020). Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-kappaB Innate Immune Response Transcription Factor Relish. Genetics. PubMed ID: 33109529
Summary:
Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by NF-κB transcription factors. Using a Drosophila melanogaster TBI model, previous work found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. This study found by mass spectrometry that Rel protein level increased in fly heads at 4-8 h after TBI. To investigate the necessity of Rel for secondary injuries, a null allele, Rel(del), was generated by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Rel(del) mutation reduced mortality at 24 h after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Rel(del) on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Rel(del) on TBI outcomes, genome-wide mRNA expression profiles of uninjured and injured +/+, +/Rel(del) , and Rel(del) /Rel(del) flies were compared at 4 h following TBI. Only a few genes changed expression more than two-fold in +/Rel(del) flies relative to +/+ and Rel(del) /Rel(del) flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

Xu, W., Xie, X. J., Faust, A. K., Liu, M., Li, X., Chen, F., Naquin, A. A., Walton, A. C., Kishbaugh, P. W. and Ji, J. Y. (2020). All-Atomic Molecular Dynamic Studies of Human and Drosophila CDK8: Insights into Their Kinase Domains, the LXXLL Motifs, and Drug Binding Site. Int J Mol Sci 21(20). PubMed ID: 33053834
Summary:
Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, the structures of human and Drosophila CDK8 proteins were analyzed using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, the structural differences were evaluated between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, it was observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, the inhibitor-induced conformational changes of hCDK8 were identified, and the results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.

Won, J. H. and Cho, K. O. (2020). Wg secreted by conventional Golgi transport diffuses and forms Wg gradient whereas Wg tethered to extracellular vesicles do not diffuse. Cell Death Differ. PubMed ID: 33028960
Summary:
Wingless (Wg)/Wnt family proteins are essential for animal development and adult homeostasis. Drosophila Wg secreted from the dorsal-ventral (DV) midline in wing discs forms a concentration gradient that is shaped by diffusion rate and stability of Wg. To understand how the gradient of extracellular Wg is generated, the secretion route of NRT-Wg, an artificial membrane-tethered form of Wg that is supposedly not secreted but still supports fly development, was compared to that of wild-type Wg. Wild-type Wg is secreted by both conventional Golgi transport and via extracellular vesicles (EVs), and NRT-Wg can be also secreted via EVs. Furthermore, wild-type Wg secreted by Golgi transport diffused and formed Wg gradient but Wg-containing EVs did not diffuse at all. In case of Wg stability, Sol narae (Sona), a metalloprotease that cleaves Wg, contributes to generate a steep Wg gradient. Interestingly, Wg was also produced in the presumptive wing blade region, which indicates that NRT-Wg on EVs expressed in the blade allows the blade cells to proliferate and differentiate without Wg diffused from the DV midline. It is proposed that EV-associated Wg induces Wg signaling in autocrine and juxtaposed manners whereas Wg secreted by Golgi transport forms gradient and acts in the long-range signaling, and different organs differentially utilize these two types of Wg signaling for their own development.

Terriente-Felix, A., Wilson, E. L. and Whitworth, A. J. (2020). Drosophila phosphatidylinositol-4 kinase fwd promotes mitochondrial fission and can suppress Pink1/parkin phenotypes. PLoS Genet 16(10): e1008844. PubMed ID: 33085661
Summary:
Balanced mitochondrial fission and fusion play an important role in shaping and distributing mitochondria, as well as contributing to mitochondrial homeostasis and adaptation to stress. In particular, mitochondrial fission is required to facilitate degradation of damaged or dysfunctional units via mitophagy. Two Parkinson's disease factors, PINK1 and Parkin, are considered key mediators of damage-induced mitophagy, and promoting mitochondrial fission is sufficient to suppress the pathological phenotypes in Drosophila Pink1/parkin mutants. Additional factors were sought that impinge on mitochondrial dynamics and which may also suppress Pink1/parkin phenotypes. The Drosophila phosphatidylinositol 4-kinase IIIβ homologue, Four wheel drive (Fwd), promotes mitochondrial fission downstream of the pro-fission factor Drp1. Previously described only as male sterile, this study identified several new phenotypes in fwd mutants, including locomotor deficits and shortened lifespan, which are accompanied by mitochondrial dysfunction. Finally, fwd overexpression can suppress locomotor deficits and mitochondrial disruption in Pink1/parkin mutants, consistent with its function in promoting mitochondrial fission. Together these results shed light on the complex mechanisms of mitochondrial fission and further underscore the potential of modulating mitochondrial fission/fusion dynamics in the context of neurodegeneration.

Bairzin, J. C. D., Emmons-Bell, M. and Hariharan, I. K. (2020). The Hippo pathway coactivator Yorkie can reprogram cell fates and create compartment-boundary-like interactions at clone margins. Sci Adv 6(50). PubMed ID: 33298454
Summary:
During development, tissue-specific patterns of gene expression are established by transcription factors and then stably maintained via epigenetic mechanisms. Cancer cells often express genes that are inappropriate for that tissue or developmental stage. This study shows that high activity levels of Yki, the Hippo pathway coactivator that causes overgrowth in Drosophila imaginal discs, can also disrupt cell fates by altering expression of selector genes like engrailed (en) and Ultrabithorax (Ubx). Posterior clones expressing activated Yki can down-regulate en and express an anterior selector gene, cubitus interruptus (ci). The microRNA bantam and the chromatin regulator Taranis both function downstream of Yki in promoting ci expression. The boundary between Yki-expressing posterior clones and surrounding wild-type cells acquires properties reminiscent of the anteroposterior compartment boundary; Hedgehog signaling pathway activation results in production of Dpp. Thus, at least in principle, heterotypic interactions between Yki-expressing cells and their neighbors could activate boundary-specific signaling mechanisms.

Zhang, T., Periz, G., Lu, Y. N. and Wang, J. (2020). USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway. Proc Natl Acad Sci U S A 117(45): 28114-28125. PubMed ID: 33106424
Summary:
An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). This study reports the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.

Wednesday, January 20th - Adult neural development and function

Wang, Y. H., Ding, Z. Y., Cheng, Y. J., Chien, C. T. and Huang, M. L. (2020). An Efficient Screen for Cell-Intrinsic Factors Identifies the Chaperonin CCT and Multiple Conserved Mechanisms as Mediating Dendrite Morphogenesis. Front Cell Neurosci 14: 577315. PubMed ID: 33100975
Summary:
Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. This study conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. CCT is shown to localize in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. This study also reveals novel mechanisms underlying dendrite morphogenesis. For example, it was shown that components of Drosophila Nogo signaling (POSH and Tango14), are required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. This methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.

Aboukilila, M. Y., Sami, J. D., Wang, J., England, W., Spitale, R. C. and Cleary, M. D. (2020). Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling. PLoS One 15(12): e0240386. PubMed ID: 33264304
Summary:
Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. This study sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Aims in this study were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on "mock tagged" samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.

Oroz, J., Felix, S. S., Cabrita, E. J. and Laurents, D. V. (2020). Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation. J Biol Chem. PubMed ID: 33093173
Summary:
The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. This study has characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on (13)C detection to afford an essentially complete set of (13)Cα, (13)Cβ, (1)Hα and backbone (13)CO and (15)N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55-60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca(++), but do bind Zn(++), which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.

Agrawal, S., Dickinson, E. S., Sustar, A., Gurung, P., Shepherd, D., Truman, J. W. and Tuthill, J. C. (2020). Central processing of leg proprioception in Drosophila. Elife 9. PubMed ID: 33263281
Summary:
Proprioception, the sense of self-movement and position, is mediated by mechanosensory neurons that detect diverse features of body kinematics. Although proprioceptive feedback is crucial for accurate motor control, little is known about how downstream circuits transform limb sensory information to guide motor output. This study investigated neural circuits in Drosophila that process proprioceptive information from the fly leg. Three cell types from distinct developmental lineages were identified that are positioned to receive input from proprioceptor subtypes encoding tibia position, movement, and vibration. 13Bα neurons encode femur-tibia joint angle and mediate postural changes in tibia position. 9Aα neurons also drive changes in leg posture, but encode a combination of directional movement, high frequency vibration, and joint angle. Activating 10Bα neurons, which encode tibia vibration at specific joint angles, elicits pausing in walking flies. Altogether, these results reveal that central circuits integrate information across proprioceptor subtypes to construct complex sensorimotor representations that mediate diverse behaviors, including reflexive control of limb posture and detection of leg vibration.

Sun, Y., Qiu, R., Li, X., Cheng, Y., Gao, S., Kong, F., Liu, L. and Zhu, Y. (2020). Social attraction in Drosophila is regulated by the mushroom body and serotonergic system. Nat Commun 11(1): 5350. PubMed ID: 33093442
Summary:
Sociality is among the most important motivators of human behaviour. However, the neural mechanisms determining levels of sociality are largely unknown, primarily due to a lack of suitable animal models. This study reports the presence of a surprising degree of general sociality in Drosophila. A newly-developed paradigm to study social approach behaviour in flies reveal that social cues perceive through both vision and olfaction converged in a central brain region, the γ lobe of the mushroom body, which exhibite activation in response to social experience. The activity of these γ neurons control the motivational drive for social interaction. At the molecular level, the serotonergic system is critical for social affinity. These results demonstrate that Drosophila are highly sociable, providing a suitable model system for elucidating the mechanisms underlying the motivation for sociality.

Sharkey, C. R., Blanco, J., Leibowitz, M. M., Pinto-Benito, D. and Wardill, T. J. (2020). The spectral sensitivity of Drosophila photoreceptors. Sci Rep 10(1): 18242. PubMed ID: 33106518
Summary:
Drosophila melanogaster has long been a popular model insect species, due in large part to the availability of genetic tools and is fast becoming the model for insect colour vision. Key to understanding colour reception in Drosophila is in-depth knowledge of spectral inputs and downstream neural processing. While recent studies have sparked renewed interest in colour processing in Drosophila, photoreceptor spectral sensitivity measurements have yet to be carried out in vivo. This study has fully characterised the spectral input to the motion and colour vision pathways, and directly measured the effects of spectral modulating factors, screening pigment density and carotenoid-based ocular pigments. All receptor sensitivities had significant shifts in spectral sensitivity compared to previous measurements. Notably, the spectral range of the Rh6 visual pigment is substantially broadened and its peak sensitivity is shifted by 92 nm from 508 to 600 nm. This deviation can be explained by transmission of long wavelengths through the red screening pigment and by the presence of the blue-absorbing filter in the R7y receptors. Further, direct interactions between inner and outer photoreceptors were tested using selective recovery of activity in photoreceptor pairs.

Tuesday, December 19th - Stem cells

Shard, C., Luna-Escalante, J. and Schweisguth, F. (2020). Tissue-wide coordination of epithelium-to-neural stem cell transition in the Drosophila optic lobe requires Neuralized. J Cell Biol 219(11). PubMed ID: 32946560
Summary:
Many tissues are produced by specialized progenitor cells emanating from epithelia via epithelial-to-mesenchymal transition (EMT). Most studies have so far focused on EMT involving single or isolated groups of cells. This study describes an EMT-like process that requires tissue-level coordination. This EMT-like process occurs along a continuous front in the Drosophila optic lobe neuroepithelium to produce neural stem cells (NSCs). Emerging NSCs remain epithelial and apically constrict before dividing asymmetrically to produce neurons. Apical constriction is associated with contractile myosin pulses and involves RhoGEF3 and down-regulation of the Crumbs complex by the E3 ubiquitin ligase Neuralized. Anisotropy in Crumbs complex levels also results in accumulation of junctional myosin. Disrupting the regulation of Crumbs by Neuralized lowered junctional myosin and led to imprecision in the integration of emerging NSCs into the front. Thus, Neuralized promotes smooth progression of the differentiation front by coupling epithelium remodeling at the tissue level with NSC fate acquisition.

Mehrotra, S., Bansal, P., Oli, N., Pillai, S. J. and Galande, S. (2020). Defective Proventriculus Regulates Cell Specification in the Gastric Region of Drosophila Intestine. Front Physiol 11: 711. PubMed ID: 32760283
Summary:
The adult Drosophila midgut and the mammalian intestine share a high degree of conservation between such signaling pathways. The gastric region located in the Drosophila middle-midgut coincides with the region containing fewest number of stem cells. It is also known as the copper cell (CC) region since it is composed of specialized groups of acid-secreting CCs, along with interstitial cells and enteroendocrine cells. The generation and maintenance of these cell populations are determined by the bone morphogenic protein-like Decapentaplegic (Dpp) signaling pathway. The morphogenic gradient of the Dpp signaling activity induces differential expression of specific transcription factors labial (lab) and defective proventriculus (dve), which are required for the generation of various cell types specific to this region. This study investigated the role of Dve in regulation of tissue homeostasis in the CC region. These studies reveal that ectopic expression of dve in stem cells suppresses their self-renewal throughout the intestine. It was further demonstrated that Dve is not required for generation of CCs. Higher levels of Dve can alter cell specification by inhibition of cut expression, which in turn prevents CC formation during homeostasis.

Yoshinari, Y., Ameku, T., Kondo, S., Tanimoto, H., Kuraishi, T., Shimada-Niwa, Y. and Niwa, R. (2020). Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster. Elife 9. PubMed ID: 33077027
Summary:
Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. This study reports that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca(2+) signaling. Moreover, Matrix metalloproteinase-2 was identified as a downstream component of the octopamine-Ca(2+) signaling to induce GSC increase. This study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling.

Arthurton, L., Nahotko, D. A., Alonso, J., Wendler, F. and Baena-Lopez, L. A. (2020). Non-apoptotic caspase activation preserves Drosophila intestinal progenitor cells in quiescence. EMBO Rep: e48892. PubMed ID: 33135280
Summary:
Caspase malfunction in stem cells often precedes the appearance and progression of multiple types of cancer, including human colorectal cancer. However, the caspase-dependent regulation of intestinal stem cell properties remains poorly understood. This study demonstrates that Dronc, the Drosophila ortholog of caspase-9/2 in mammals, limits the number of intestinal progenitor cells and their entry into the enterocyte differentiation programme. Strikingly, these unexpected roles for Dronc are non-apoptotic and have been uncovered under experimental conditions without epithelial replenishment. Supporting the non-apoptotic nature of these functions, this study shows that they require the enzymatic activity of Dronc, but are largely independent of the apoptotic pathway. Alternatively, the genetic and functional data suggest that they are linked to the caspase-mediated regulation of Notch signalling. These findings provide novel insights into the non-apoptotic, caspase-dependent modulation of stem cell properties that could improve understanding of the origin of intestinal malignancies.

Melamed, D. and Kalderon, D. (2020). Opposing JAK-STAT and Wnt signaling gradients define a stem cell domain by regulating differentiation at two borders. Elife 9. PubMed ID: 33135631
Summary:
Many adult stem cell communities are maintained by population asymmetry, where stochastic behaviors of multiple individual cells collectively result in a balance between stem cell division and differentiation. This study investigated how this is achieved for Drosophila Follicle Stem Cells (FSCs) by spatially-restricted niche signals. FSCs produce transit-amplifying Follicle Cells (FCs) from their posterior face and quiescent Escort Cells (ECs) to their anterior. JAK-STAT pathway activity, which declines from posterior to anterior, dictates the pattern of divisions over the FSC domain, promotes more posterior FSC locations and conversion to FCs, while opposing EC production. Wnt pathway activity declines from the anterior, promotes anterior FSC locations and EC production, and opposes FC production. The pathways combine to define a stem cell domain through concerted effects on FSC differentiation to ECs and FCs at either end of opposing signaling gradients, and impose a pattern of proliferation that matches derivative production.

Sharma, A., Akagi, K., Pattavina, B., Wilson, K. A., Nelson, C., Watson, M., Maksoud, E., Harata, A., Ortega, M., Brem, R. B. and Kapahi, P. (2020). Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal permeability and survival in Drosophila. Sci Rep 10(1): 19080. PubMed ID: 33154387
Summary:
Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. This study developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) was performed to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, an RNA binding protein, Musashi (msi), was identified as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, this study has established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.

Monday, January 18th - Enhancers and gene regulation

Lopez-Rivera, F., Foster Rhoades, O. K., Vincent, B. J., Pym, E. C. G., Bragdon, M. D. J., Estrada, J., DePace, A. H. and Wunderlich, Z. (2020). A Mutation in the Drosophila melanogaster eve Stripe 2 Minimal Enhancer Is Buffered by Flanking Sequences. G3 (Bethesda). PubMed ID: 33037064
Summary:
Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene's endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. It is hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogaster even-skipped stripe 2 enhancer as a case study, the effect was examined of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. In contrast to the misexpression caused by the gt-2 binding site deletion in the minimal enhancer, the same gt-2 binding site deletion in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Deleting the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. These results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.

Ogienko, A. A., Andreyeva, E. N., Omelina, E. S., Oshchepkova, A. L. and Pindyurin, A. V. (2020). Molecular and cytological analysis of widely-used Gal4 driver lines for Drosophila neurobiology. BMC Genet 21(Suppl 1): 96. PubMed ID: 33092520
Summary:
The Drosophila central nervous system (CNS) is a convenient model system for the study of the molecular mechanisms of conserved neurobiological processes. The manipulation of gene activity in specific cell types and subtypes of the Drosophila CNS is frequently achieved by employing the binary Gal4/UAS system. However, many Gal4 driver lines available from the Bloomington Drosophila Stock Center (BDSC) and commonly used in Drosophila neurobiology are still not well characterized. Among these are three lines with Gal4 driven by the repo promoter (BDSC #7415), and the 69B-Gal4 line (BDSC #1774). For most of these lines, the exact insertion sites of the transgenes and the detailed expression patterns of Gal4 are not known. This study is aimed at filling these gaps. This study has mapped the genomic location of the Gal4-bearing P-elements carried by the BDSC lines #8760, #8765, #458, #7415, and #1774. In addition, for each of these lines, the Gal4-driven GFP expression pattern was analyzed in the third instar larval CNS and eye-antennal imaginal discs. Localizations of the endogenous Elav and Repo proteins were used as markers of neuronal and glial cells, respectively. This study provides a mini-atlas of the spatial activity of Gal4 drivers that are widely used for the expression of UAS-target genes in the Drosophila CNS. The data will be helpful for planning experiments with these drivers and for the correct interpretation of the results.

Islam, I. M., Ng, J., Valentino, P. and Erclik, T. (2020). Identification of enhancers that drive the spatially restricted expression of Vsx1 and Rx in the outer proliferation center of the developing Drosophila optic lobe. Genome: 1-9. PubMed ID: 33054400
Summary:
Combinatorial spatial and temporal patterning of stem cells is a powerful mechanism for the generation of neural diversity in insect and vertebrate nervous systems. In the developing Drosophila medulla, the neural stem cells of the outer proliferation center (OPC) are spatially patterned by the mutually exclusive expression of three homeobox transcription factors: Vsx1 in the center of the OPC crescent (cOPC), Optix in the main arms (mOPC), and Rx in the posterior tips (pOPC). These spatial factors act together with a temporal cascade of transcription factors in OPC neuroblasts to specify the greater than 80 medulla cell types. This study identified the enhancers that are sufficient to drive the spatially restricted expression of the Vsx1 and Rx genes in the OPC. Removal of the cOPC enhancer in the Muddled inversion mutant leads to the loss of Vsx1 expression in the cOPC. Analysis of the evolutionarily conserved sequences within these enhancers suggests that direct repression by Optix may restrict the expression of Vsx1 and Rx to the cOPC and pOPC, respectively.

Weaver, L. N. and Drummond-Barbosa, D. (2020). The Nuclear Receptor Seven Up Regulates Genes Involved in Immunity and Xenobiotic Response in the Adult Drosophila Female Fat Body. G3 (Bethesda). PubMed ID: 33087412
Summary:
There is increasing evidence that nuclear receptor signaling in peripheral tissues can influence oogenesis. It was previously shown that the Drosophila nuclear receptor Seven up (Svp) is required in the adult fat body to regulate distinct steps of oogenesis; however, the relevant downstream targets of Svp remain unknown. This study took an RNA sequencing approach to identify candidate Svp targets specifically in the adult female fat body that might mediate this response. svp knockdown in the adult female fat body significantly downregulated immune genes involved in the first line of pathogen defense, suggesting a role for Svp in stimulating early immunity. In addition, it was found that Svp transcriptionally regulates genes involved in each step of the xenobiotic detoxification response. Based on these findings, a testable model is proposed in which Svp functions in the adult female fat body to stimulate early defense against pathogens and facilitate detoxification as part of its mechanisms to promote oogenesis.

Berrocal, A., Lammers, N. C., Garcia, H. G. and Eisen, M. B. (2020). Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene. Elife 9. PubMed ID: 33300492
Summary:
Live imaging was used to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its seven stripe expression pattern forms, and tools were developed to characterize and visualize how transcriptional bursting varies over time and space. Despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, this study shows that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. This work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shape complex developmental patterns.

Fuqua, T., Jordan, J., van Breugel, M. E., Halavatyi, A., Tischer, C., Polidoro, P., Abe, N., Tsai, A., Mann, R. S., Stern, D. L. and Crocker, J. (2020). Dense and pleiotropic regulatory information in a developmental enhancer. Nature 587(7833): 235-239. PubMed ID: 33057197
Summary:
Changes in gene regulation underlie much of phenotypic evolution. However, understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, this study surveyed an unbiased mutation library for a developmental enhancer from the shavenbaby (svb; also known as ovo) gene in Drosophila melanogaster. Almost all mutations were found to alter gene expression, and parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

Friday, January 15th - Adult Development

Tan, H., Fulton, R. E., Chou, W. H., Birkholz, D. A., Mannino, M. P., Yamaguchi, D. M., Aldrich, J. C., Jacobsen, T. L. and Britt, S. G. (2020). Drosophila R8 photoreceptor cell subtype specification requires hibris. PLoS One 15(10): e0240451. PubMed ID: 33052948
Summary:
Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. A genetic screen was conducted to identify additional regulators of this process and a novel mutation in the hibris gene, which encodes an irre cell recognition module protein (IRM), was identified. These immunoglobulin super family cell adhesion molecules include human KIRREL and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptor cell diversity. This study demonstrates that hibris is required within the retina, and that hibris over-expression is sufficient to disrupt normal photoreceptor cell patterning. These findings suggest an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells.

Rice, C., Macdonald, S. J., Wang, X. and Ward, R. E. (2020). The Broad Transcription Factor Links Hormonal Signaling, Gene Expression and Cellular Morphogenesis Events During Drosophila Imaginal Disc Development. Genetics. PubMed ID: 33115752
Summary:
Imaginal disc morphogenesis during metamorphosis in Drosophila melanogaster provides an excellent model to uncover molecular mechanisms by which hormonal signals effect physical changes during development. The broad (br) Z2 isoform encodes a transcription factor required for disc morphogenesis in response to 20-hydroxyecdysone, yet how it accomplishes this remains largely unknown. Thus study use functional studies of amorphic br(5) mutants and a transcriptional target approach to identify processes driven by br and its regulatory targets in leg imaginal discs. br(5) mutants fail to properly remodel their basal extracellular matrix (ECM) between 4 and 7 hours after puparium formation. Additionally, br(5) mutant discs do not undergo the cell shape changes necessary for leg elongation and fail to elongate normally when exposed to the protease trypsin. RNA sequencing of wild type and br(5) mutant leg discs identified 717 genes differentially regulated by br, including a large number of genes involved in glycolysis, and genes that encode proteins that interact with the ECM. RNAi-based functional studies reveal that several of these genes are required for adult leg formation, particularly those involved in remodeling the ECM. Additionally, br Z2 expression is abruptly shut down at the onset of metamorphosis, and expressing it beyond this time results in failure of leg development during the late prepupal and pupal stages. Taken together, these results suggest that br Z2 is required to drive ECM remodeling, change cell shape, and maintain metabolic activity through the mid prepupal stage, but must be switched off to allow expression of pupation genes.

Hebbar, S., Schuhmann, K., Shevchenko, A. and Knust, E. (2020). Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis. J Cell Biol 219(12). PubMed ID: 33048164
Summary:
Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. This study addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to understanding of apical growth during morphogenesis.

Vasquez-Procopio, J., Rajpurohit, S. and Missirlis, F. (2020). Cuticle darkening correlates with increased body copper content in Drosophila melanogaster. Biometals 33(6): 293-303. PubMed ID: 33026606
Summary:
Insect epidermal cells secrete a cuticle that serves as an exoskeleton providing mechanical rigidity to each individual, but also insulation, camouflage or communication within their environment. Cuticle deposition and hardening (sclerotization) and pigment synthesis are parallel processes requiring tyrosinase activity, which depends on an unidentified copper-dependent enzyme component in Drosophila melanogaster. This study determined the metallomes of fly strains selected for lighter or darker cuticles in a laboratory evolution experiment, asking whether any specific element changed in abundance in concert with pigment deposition. The results showed a correlation between total iron content and strength of pigmentation, which was further corroborated by ferritin iron quantification. To ask if the observed increase in iron body content along with increased pigment deposition could be generalizable, yellow and ebony alleles causing light and dark pigmentation, respectively, were crossed into similar genetic backgrounds and their metallomes were measured. Iron remained unaffected in the various mutants providing no support for a causative link between pigmentation and iron content. In contrast, the combined analysis of both experiments suggested instead a correlation between pigment deposition and total copper body content, possibly due to increased demand for epidermal tyrosinase activity.

Peterson, N. G., Stormo, B. M., Schoenfelder, K. P., King, J. S., Lee, R. R. and Fox, D. T. (2020). Cytoplasmic sharing through apical membrane remodeling. Elife 9. PubMed ID: 33051002
Summary:
Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, this study discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 62 kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as incomplete cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins Ogre (Inx1), Inx2, and Inx3 (normally associated with transport of molecules < 1 kDa), which are positioned by membrane remodeling GTPases. This work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.

Csordas, G., Grawe, F. and Uhlirova, M. (2020). Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments. Elife 9. PubMed ID: 33026342
Summary:
Blood development in multicellular organisms relies on specific tissue microenvironments that nurture hematopoietic precursors and promote their self-renewal, proliferation, and differentiation. The mechanisms driving blood cell homing and their interactions with hematopoietic microenvironments remain poorly understood. This study use the Drosophila melanogaster model to reveal a pivotal role for basement membrane composition in the formation of hematopoietic compartments. By modulating extracellular matrix components, the fly blood cells known as hemocytes can be relocated to tissue surfaces where they function similarly to their natural hematopoietic environment. This study established that the Collagen XV/XVIII ortholog Multiplexin in the tissue-basement membranes and the phagocytosis receptor Eater on the hemocytes physically interact and are necessary and sufficient to induce immune cell-tissue association. These results highlight the cooperation of Multiplexin and Eater as an integral part of a homing mechanism that specifies and maintains hematopoietic sites in Drosophila.

Thursday, January 14th - Gonads

Ogienko, A. A., Yarinich, L. A., Fedorova, E. V., Dorogova, N. V., Bayborodin, S. I., Baricheva, E. M. and Pindyurin, A. V. (2020). GAGA Regulates Border Cell Migration in Drosophila. Int J Mol Sci 21(20). PubMed ID: 33050455
Summary:
Collective cell migration is a complex process that happens during normal development of many multicellular organisms, as well as during oncological transformations. In Drosophila oogenesis, a small set of follicle cells originally located at the anterior tip of each egg chamber become motile and migrate as a cluster through nurse cells toward the oocyte. These specialized cells are referred to as border cells (BCs) and provide a simple and convenient model system to study collective cell migration. The process is known to be complexly regulated at different levels and the product of the slow border cells (slbo) gene, the C/EBP transcription factor, is one of the key elements in this process. However, little is known about the regulation of slbo expression. On the other hand, the ubiquitously expressed transcription factor GAGA, which is encoded by the Trithorax-like (Trl) gene was previously demonstrated to be important for Drosophila oogenesis. This study found that Trl mutations cause substantial defects in BC migration. Partially, these defects are explained by the reduced level of slbo expression in BCs. Additionally, a strong genetic interaction between Trl and slbo mutants, along with the presence of putative GAGA binding sites within the slbo promoter and enhancer, suggests the direct regulation of this gene by GAGA. This idea is supported by the reduction in the slbo-Gal4-driven GFP expression within BC clusters in Trl mutant background. However, the inability of slbo overexpression to compensate defects in BC migration caused by Trl mutations suggests that there are other GAGA target genes contributing to this process. Taken together, the results define GAGA as another important regulator of BC migration in Drosophila oogenesis.

Rockwell, A. L. and Hongay, C. F. (2020). Dm Ime4 depletion affects permeability barrier and chic function in Drosophila spermatogenesis. Mech Dev: 103650. PubMed ID: 33038528
Summary:
Adenosine methylation of messenger RNA at the N(6) position (m(6)A) is a non-editing modification that can affect several aspects of mRNA metabolism. Dm Ime4, also known as METTL3, MTA, and MTA-70 in other organisms, is the catalytic subunit of the methyltransferase complex that adds this modification. Using a strategy that depletes Dm Ime4 specifically in the somatic cyst cells of Drosophila testes without affecting essential functions in development, this study has found that Dm Ime4 may potentially regulate splicing of profilin (chic) mRNA, the message for an essential and evolutionarily conserved protein mainly known for its function in actin polymerization. One of the lesser known roles for Chic is its requirement for establishment and maintenance of the somatic cyst-cell permeability barrier in Drosophila spermatogenesis. Chic and Dm Ime4 colocalize and are abundant in somatic cyst cells throughout spermatogenesis. Upon selective depletion of Dm Ime4, significant reduction of Chic protein levels and malfunction of the permeability barrier were observed. chic mRNA contains intronic Ime4 binding sites that can form the hairpin structures required for recognition by the methyltransferase complex. These data show that the reduced levels of Chic protein observed in ime4 somatic cyst-cell knockdowns could be the result of aberrant splicing of its mRNA. In turn, low levels of Chic are known to affect the function of the somatic permeability barrier, leading to germline death and the reduced fertility observed in ime4 knockdown males. It is proposed that Ime4 may regulate chic in other developmental contexts and in other organisms, including mice and humans. Chic is an essential protein that is evolutionarily conserved, and establishment and maintenance of cell barriers and domains are important strategies used in metazoan development. Taken together, these findings define a framework to investigate specific functions of Ime4 and its homologs in multicellular organisms by bypassing its pleiotropic requirement in early developmental stages.

Sato, K., Ito, H. and Yamamoto, D. (2020). teiresias, a Fruitless target gene encoding an immunoglobulin-superfamily transmembrane protein, is required for neuronal feminization in Drosophila. Commun Biol 3(1): 598. PubMed ID: 33087851
Summary:
This study aims at identifying transcriptional targets of FruitlessBM (FruBM), which represents the major isoform of male-specific FruM transcription factors that induce neural sexual dimorphisms. A promoter of the axon-guidance factor gene robo1 carries the 16-bp palindrome motif Pal1, to which FruM binds. A genome-wide search for Pal1-homologous sequences yielded ~200 candidate genes. Among these, CG17716 potentially encodes a transmembrane protein with extracellular immunoglobulin (Ig)-like domains similar to Robo1. Indeed, FruBM overexpression reduced CG17716 mRNA and protein expression. In the fru-expressing mAL neuron cluster exhibiting sexual dimorphism, this study found that CG17716 knockdown in female neurons completely transformed all neurites to the male-type. Conversely, CG17716 overexpression suppressed male-specific midline crossing of fru-expressing sensory axons. CG17716 was renamed teiresias (tei) based on this feminizing function. It is hypothesize that Tei interacts with other Ig superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females, whereas FruBM represses tei transcription in males.

Stevens, C. A., Revaitis, N. T., Caur, R. and Yakoby, N. (2020). The ETS-transcription factor Pointed is sufficient to regulate the posterior fate of the follicular epithelium. Development 147(22). PubMed ID: 33028611
Summary:
The Janus-kinase/signal transducer and activator of transcription (JAK/STAT) pathway regulates the anterior posterior axis of the Drosophila follicle cells. In the anterior, it activates the bone morphogenetic protein (BMP) signaling pathway through expression of the BMP ligand decapentaplegic (dpp). In the posterior, JAK/STAT works with the epidermal growth factor receptor (EGFR) pathway to express the T-box transcription factor midline (mid). Although MID is necessary for establishing the posterior fate of the egg chamber, this study shows that it is not sufficient to determine a posterior fate. The ETS-transcription factor pointed (pnt) is expressed in an overlapping domain to mid in the follicle cells. This study shows that pnt is upstream of mid and that it is sufficient to induce a posterior fate in the anterior end, which is characterized by the induction of mid, the prevention of the stretched cells formation and the abrogation of border cell migration. It is demonstrated that the anterior BMP signaling is abolished by PNT through dpp repression. However, ectopic DPP cannot rescue the anterior fate formation, suggesting additional targets of PNT participate in the posterior fate determination.

Kaufman, R. S., Price, K. L., Mannix, K. M., Ayers, K. M., Hudson, A. M. and Cooley, L. (2020). Drosophila sperm development and intercellular cytoplasm sharing through ring canals do not require an intact fusome. Development 147(22). PubMed ID: 33033119
Summary:
Animal germ cells communicate directly with each other during gametogenesis through intercellular bridges, often called ring canals (RCs), that form as a consequence of incomplete cytokinesis during cell division. Developing germ cells in Drosophila have an additional specialized organelle connecting the cells called the fusome. Ring canals and the fusome are required for fertility in Drosophila females, but little is known about their roles during spermatogenesis. With live imaging, this study directly observed the intercellular movement of GFP and a subset of endogenous proteins through RCs during spermatogenesis, from two-cell diploid spermatogonia to clusters of 64 post-meiotic haploid spermatids, demonstrating that RCs are stable and open to intercellular traffic throughout spermatogenesis. Disruption of the fusome, a large cytoplasmic structure that extends through RCs and is important during oogenesis, had no effect on spermatogenesis or male fertility under normal conditions. These results reveal that male germline RCs allow the sharing of cytoplasmic information that might play a role in quality control surveillance during sperm development.

Osman, I. and Pek, J. W. (2020). Maternally inherited intron coordinates primordial germ cell homeostasis during Drosophila embryogenesis. Cell Death Differ. PubMed ID: 33093656
Summary:
Primordial germ cells (PGCs) give rise to the germline stem cells (GSCs) in the adult Drosophila gonads. Both PGCs and GSCs need to be tightly regulated to safeguard the survival of the entire species. During larval development, a non-cell autonomous homeostatic mechanism is in place to maintain PGC number in the gonads. Whether such germline homeostasis occurs during early embryogenesis before PGCs reach the gonads remains unclear. Previous work has shown that the maternally deposited sisRNA sisR-2 can influence GSC number in the female progeny. This study uncover the presence of a homeostatic mechanism regulating PGCs during embryogenesis. sisR-2 represses PGC number by promoting PGC death. Surprisingly, increasing maternal sisR-2 leads to an increase in PGC death, but no drop in PGC number was observed. This is due to ectopic division of PGCs via the de-repression of Cyclin B, which is governed by a genetic pathway involving sisR-2, bantam and brat. A cell autonomous model is proposed whereby germline homeostasis is achieved by preserving PGC number during embryogenesis.

Wednesday, January 13th - Signaling

Raj, A., Chimata, A. V. and Singh, A. (2020). Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila. Sci Rep 10(1): 17221. PubMed ID: 33057115
Summary:
The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. This study found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wg(RNAi), shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. Upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. These data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development.

Panta, M., Kump, A. J., Dalloul, J. M., Schwab, K. R. and Ahmad, S. M. (2020). Three distinct mechanisms, Notch instructive, permissive, and independent, regulate the expression of two different pericardial genes to specify cardiac cell subtypes. PLoS One 15(10): e0241191. PubMed ID: 33108408
Summary:
Two major cell subtypes, contractile cardial cells (CCs) and nephrocytic pericardial cells (PCs), comprise the Drosophila heart. Binding sites for Suppressor of Hairless [Su(H)], an integral transcription factor in the Notch signaling pathway, are enriched in the enhancers of PC-specific genes. Three distinct mechanisms regulating the expression of two different PC-specific genes, Holes in muscle (Him), and Zn finger homeodomain 1 (zfh1). Him transcription is activated in PCs in a permissive manner by Notch signaling: in the absence of Notch signaling, Su(H) forms a repressor complex with co-repressors and binds to the Him enhancer, repressing its transcription; upon alleviation of this repression by Notch signaling, Him transcription is activated. In contrast, zfh1 is transcribed by a Notch-instructive mechanism in most PCs, where mere alleviation of repression by preventing the binding of Su(H)-co-repressor complex is not sufficient to activate transcription. These results suggest that upon activation of Notch signaling, the Notch intracellular domain associates with Su(H) to form an activator complex that binds to the zfh1 enhancer, and that this activator complex is necessary for bringing about zfh1 transcription in these PCs. Finally, a third, Notch-independent mechanism activates zfh1 transcription in the remaining, even skipped-expressing, PCs. Collectively, these data show how the same feature, enrichment of Su(H) binding sites in PC-specific gene enhancers, is utilized by two very distinct mechanisms, one permissive, the other instructive, to contribute to the same overall goal: the specification and differentiation of a cardiac cell subtype by activation of the pericardial gene program. Furthermore, these results demonstrate that the zfh1 enhancer drives expression in two different domains using distinct Notch-instructive and Notch-independent mechanisms.

Pareek, G. and Pallanck, L. J. (2020). Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression. PLoS Genet 16(10): e1009118. PubMed ID: 33075064
Summary:
The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, a genetic approach was used in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. Null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. This work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2.

Richter, M. and Fingerhut, B. P. (2020). Regulatory Impact of the C-Terminal Tail on Charge Transfer Pathways in Drosophila Cryptochrome. Molecules 25(20). PubMed ID: 33086760
Summary:
Interconnected transcriptional and translational feedback loops are at the core of the molecular mechanism of the circadian clock. Such feedback loops are synchronized to external light entrainment by the blue light photoreceptor Cryptochrome (CRY) that undergoes conformational changes upon light absorption by an unknown photoexcitation mechanism. Light-induced charge transfer (CT) reactions in Drosophila CRY (dCRY) are investigated by state-of-the-art simulations that reveal a complex, multi-redox site nature of CT dynamics on the microscopic level. The simulations consider redox-active chromophores of the tryptophan triad (Trp triad) and further account for pathways mediated by W314 and W422 residues proximate to the C-terminal tail (CTT), thus avoiding a pre-bias to specific W-mediated CT pathways. The conducted dissipative quantum dynamics simulations employ microscopically derived model Hamiltonians and display complex and ultrafast CT dynamics on the picosecond timescale, subtly balanced by the electrostatic environment of dCRY. In silicio point mutations provide a microscopic basis for rationalizing particular CT directionality and demonstrate the degree of electrostatic control realized by a discrete set of charged amino acid residues. The predicted participation of CT states in proximity to the CTT relates the directionality of CT reactions to the spatial vicinity of a linear interaction motif. The results stress the importance of CTT directional charge transfer in addition to charge transfer via the Trp triad and call for the use of full-length CRY models including the interactions of photolyase homology region (PHR) and CTT domains.

Stapornwongkul, K. S., de Gennes, M., Cocconi, L., Salbreux, G. and Vincent, J. P. (2020). Patterning and growth control in vivo by an engineered GFP gradient. Science 370(6514): 321-327. PubMed ID: 33060356
Summary:
Morphogen gradients provide positional information during development. To uncover the minimal requirements for morphogen gradient formation, this study has engineered a synthetic morphogen in Drosophila wing primordia. An inert protein, green fluorescent protein (GFP), can form a detectable diffusion-based gradient in the presence of surface-associated anti-GFP nanobodies, which modulate the gradient by trapping the ligand and limiting leakage from the tissue. Anti-GFP nanobodies to the receptors of Dpp, a natural morphogen, were used to render them responsive to extracellular GFP. In the presence of these engineered receptors, GFP could replace Dpp to organize patterning and growth in vivo. Concomitant expression of glycosylphosphatidylinositol (GPI)-anchored nonsignaling receptors further improved patterning, to near-wild-type quality. Theoretical arguments suggest that GPI anchorage could be important for these receptors to expand the gradient length scale while at the same time reducing leakage.

Sharifkhodaei, Z. and Auld, V. J. (2020). Overexpressed Gliotactin activates BMP signaling through interfering with the Tkv-Dad association. Genome: 1-12. PubMed ID: 33064024
Summary:
Epithelial junctions ensure cell-cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli-BMP-miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3'UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. This study shows that ectopic expression of Gli interferes with Tkv-Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv-pMad signaling activity, and this effect is continuous through larval and pupal wing formation.

Tuesday, January 12th - Cytoskeleton and Junctions

Dewey, E. B., Parra, A. S. and Johnston, C. A. (2020). Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia. Sci Rep 10(1): 20165. PubMed ID: 33214581
Summary:
Epithelia are an eminent tissue type and a common driver of tumorigenesis, requiring continual precision in cell division to maintain tissue structure and genome integrity. Mitotic defects often trigger apoptosis, impairing cell viability as a tradeoff for tumor suppression. Identifying conditions that lead to cell death and understanding the mechanisms behind this response are therefore of considerable importance. Here this study investigated how epithelia of the Drosophila wing disc respond to loss of Short stop (Shot), a cytoskeletal crosslinking spectraplakin protein that was previously found to control mitotic spindle assembly and chromosome dynamics. In contrast to other known spindle-regulating genes, Shot knockdown induces apoptosis in the absence of Jun kinase (JNK) activation, but instead leads to elevated levels of active p38 kinase. Shot loss leads to double-strand break (DSB) DNA damage, and the apoptotic response is exacerbated by concomitant loss of p53. DSB accumulation is increased by suppression of the spindle assembly checkpoint, suggesting this effect results from chromosome damage during error-prone mitoses. Consistent with DSB induction, DNA damage and stress response genes, Growth arrest and DNA damage (GADD45) and Apoptosis signal-regulating kinase 1 (Ask1), were found to be transcriptionally upregulated as part of the shot-induced apoptotic response. Finally, co-depletion of Shot and GADD45 induced significantly higher rates of chromosome segregation errors in cultured cells and suppressed shot-induced mitotic arrest. These results demonstrate that epithelia are capable of mounting molecularly distinct responses to loss of different spindle-associated genes and underscore the importance of proper cytoskeletal organization in tissue homeostasis.

Pinter, R., Huber, T., Bukovics, P., Gaszler, P., Vig, A. T., Toth, M., Gazso-Gerhat, G., Farkas, D., Migh, E., Mihaly, J. and Bugyi, B. (2020). The activities of the gelsolin homology domains of flightless-I in actin dynamics. Front Mol Biosci 7: 575077. PubMed ID: 33033719
Summary:
Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I, the actin activities of Drosophila Flightless-I were studied by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. This work identifies the first three gelsolin homology domains (1-3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4-6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (~nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (~µM) monomer binding, and does not sever actin filaments. This work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4-6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1-3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. This study also showed that the gelsolin homology domains 4-6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.

Chippalkatti, R., Egger, B. and Suter, B. (2020). Mms19 promotes spindle microtubule assembly in Drosophila neural stem cells. PLoS Genet 16(11): e1008913. PubMed ID: 33211700
Summary:
Mitotic divisions depend on the timely assembly and proper orientation of the mitotic spindle. Malfunctioning of these processes can considerably delay mitosis, thereby compromising tissue growth and homeostasis, and leading to chromosomal instability. Loss of functional Mms19 drastically affects the growth and development of mitotic tissues in Drosophila larvae, and this study demonstrates that Mms19 is an important factor that promotes spindle and astral microtubule (MT) growth, and MT stability and bundling. Mms19 function is needed for the coordination of mitotic events and for the rapid progression through mitosis that is characteristic of neural stem cells. Surprisingly, Mms19 performs its mitotic activities through two different pathways. By stimulating the mitotic kinase cascade, it triggers the localization of the MT regulatory complex TACC/Msps (Transforming Acidic Coiled Coil/Minispindles, the homolog of human ch-TOG) to the centrosome. This activity of Mms19 can be rescued by stimulating the mitotic kinase cascade. However, other aspects of the Mms19 phenotypes cannot be rescued in this way, pointing to an additional mechanism of Mms19 action. This study provides evidence that Mms19 binds directly to MTs and that this stimulates MT stability and bundling.

Sechi, S., Frappaolo, A., Karimpour-Ghahnavieh, A., Fraschini, R. and Giansanti, M. G. (2020). A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila. J Cell Sci 133(21). PubMed ID: 33037125
Summary:
In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. This study demonstrates that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. These results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. It is proposed that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

Ricolo, D. and Araujo, S. J. (2020). Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching. Elife 9. PubMed ID: 33112231
Summary:
Subcellular lumen formation by single-cells involves complex cytoskeletal remodelling. Centrosomes are key players in the initiation of subcellular lumen formation in Drosophila melanogaster, but not much is known on the what leads to the growth of these subcellular luminal branches or makes them progress through a particular trajectory within the cytoplasm. This study has identified that the spectraplakin Short-stop (Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the tracheal terminal cell (TC) cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal luminal branching points in the embryonic and larval tracheal TC leading to cells with extra-subcellular lumina. These data provide the first evidence for a role for spectraplakins in single-cell lumen formation and branching.

Lopez-Gay, J. M., Nunley, H., Spencer, M., di Pietro, F., Guirao, B., Bosveld, F., Markova, O., Gaugue, I., Pelletier, S., Lubensky, D. K. and Bellaiche, Y. (2020). Apical stress fibers enable a scaling between cell mechanical response and area in epithelial tissue. Science 370(6514). PubMed ID: 33060329
Summary:
Biological systems tailor their properties and behavior to their size throughout development and in numerous aspects of physiology. However, such size scaling remains poorly understood as it applies to cell mechanics and mechanosensing. By examining how the Drosophila pupal dorsal thorax epithelium responds to morphogenetic forces, this study found that the number of apical stress fibers (aSFs) anchored to adherens junctions scales with cell apical area to limit larger cell elongation under mechanical stress. aSFs cluster Hippo pathway components, thereby scaling Hippo signaling and proliferation with area. This scaling is promoted by tricellular junctions mediating an increase in aSF nucleation rate and lifetime in larger cells. Development, homeostasis, and repair entail epithelial cell size changes driven by mechanical forces; this work highlights how, in turn, mechanosensitivity scales with cell size.

Monday, January 11th - Chromatin

Jefferies, G., Somers, J., Lohrey, I., Chaturvedi, V., Calabria, J., Marshall, O. J., Southall, T. D., Saint, R. and Murray, M. J. (2020). Maintenance of Cell Fate by the Polycomb Group Gene Sex Combs Extra Enables a Partial Epithelial Mesenchymal Transition in Drosophila. G3 (Bethesda). PubMed ID: 33051260
Summary:
Epigenetic silencing by Polycomb group (PcG) complexes can promote epithelial-mesenchymal transition (EMT) and stemness and is associated with malignancy of solid cancers. This study reports a role for Drosophila PcG repression in a partial EMT event that occurs during wing disc eversion, an early event during metamorphosis. A screen for genes required for eversion uncovered the PcG genes Sex combs extra (Sce) and Sex combs midleg (Scm). Depletion of Sce or Scm resulted in internalised wings and thoracic clefts, and loss of Sce inhibited the EMT of the peripodial epithelium and basement membrane breakdown, ex vivo. Targeted DamID (TaDa) using Dam-Pol II showed that Sce knockdown caused a genomic transcriptional response consistent with a shift towards a more stable epithelial fate. Surprisingly only 17 genes were significantly upregulated in Sce-depleted cells, including Abd-B, abd-A, caudal, and nubbin. Each of these loci were enriched for Dam-Pc binding. Of the four genes, only Abd-B was robustly upregulated in cells lacking Sce expression. RNAi knockdown of all four genes could partly suppress the Sce RNAi eversion phenotype, though Abd-B had the strongest effect. The results suggest that in the absence of continued PcG repression peripodial cells express genes such as Abd-B, which promote epithelial state and thereby disrupt eversion. These results emphasise the important role that PcG suppression can play in maintaining cell states required for morphogenetic events throughout development and suggest that PcG repression of Hox genes may affect epithelial traits that could contribute to metastasis.

Saha, P., Sowpati, D. T., Soujanya, M., Srivastava, I. and Mishra, R. K. (2020). Interplay of pericentromeric genome organization and chromatin landscape regulates the expression of Drosophila melanogaster heterochromatic genes. Epigenetics Chromatin 13(1): 41. PubMed ID: 33028366
Summary:
Transcription of genes residing within constitutive heterochromatin is paradoxical to the tenets of epigenetic code. The regulatory mechanisms of Drosophila melanogaster heterochromatic gene transcription remain largely unknown. Emerging evidence suggests that genome organization and transcriptional regulation are inter-linked. However, the pericentromeric genome organization is relatively less studied. Therefore, this study sought to characterize the pericentromeric genome organization and understand how this organization along with the pericentromeric factors influences heterochromatic gene expression. This study characterized the pericentromeric genome organization in Drosophila melanogaster using 5C sequencing. Heterochromatic topologically associating domains (Het TADs) correlate with distinct epigenomic domains of active and repressed heterochromatic genes at the pericentromeres. These genes are known to depend on the heterochromatic landscape for their expression. However, HP1a or Su(var)3-9 RNAi has minimal effects on heterochromatic gene expression, despite causing significant changes in the global Het TAD organization. Probing further into this observation, this study reports the role of two other chromatin proteins enriched at the pericentromeres-dMES-4 and dADD1 in regulating the expression of a subset of heterochromatic genes. It is concluded that distinct pericentromeric genome organization and chromatin landscapes maintained by the interplay of heterochromatic factors (HP1a, H3K9me3, dMES-4 and dADD1) are sufficient to support heterochromatic gene expression despite the loss of global Het TAD structure. These findings open new avenues for future investigations into the mechanisms of heterochromatic gene expression.

Galan, S., Machnik, N., Kruse, K., Diaz, N., Marti-Renom, M. A. and Vaquerizas, J. M. (2020). CHESS enables quantitative comparison of chromatin contact data and automatic feature extraction. Nat Genet 52(11): 1247-1255. PubMed ID: 33077914
Summary:
Dynamic changes in the three-dimensional (3D) organization of chromatin are associated with central biological processes, such as transcription, replication and development. Therefore, the comprehensive identification and quantification of these changes is fundamental to understanding of evolutionary and regulatory mechanisms. This study presents Comparison of Hi-C Experiments using Structural Similarity (CHESS), an algorithm for the comparison of chromatin contact maps and automatic differential feature extraction. The robustness of CHESS to experimental variability is presented, and its biological applications were showcased on (1) interspecies comparisons of syntenic regions in human and mouse models; (2) intraspecies identification of conformational changes in Zelda-depleted Drosophila embryos; (3) patient-specific aberrant chromatin conformation in a diffuse large B-cell lymphoma sample; and (4) the systematic identification of chromatin contact differences in high-resolution Capture-C data. In summary, CHESS is a computationally efficient method for the comparison and classification of changes in chromatin contact data.

Mahesh, G., Rivas, G. B. S., Caster, C., Ost, E. B., Amunugama, R., Jones, R., Allen, D. L. and Hardin, P. E. (2020). Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A. Sci Rep 10(1): 17951. PubMed ID: 33087840
Summary:
Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. This study used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB; see Non-stop) module lengthened period similarly to Nipped-A RNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.

Jain, S. U., Rashoff, A. Q., Krabbenhoft, S. D., Hoelper, D., Do, T. J., Gibson, T. J., Lundgren, S. M., Bondra, E. R., Deshmukh, S., Harutyunyan, A. S., Juretic, N., Jabado, N., Harrison, M. M. and Lewis, P. W. (2020). H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2. Mol Cell 80(4): 726-735. PubMed ID: 33049227
Summary:
Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M; see Drosophila H3) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. This study reports that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. These results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

Eck, E., Liu, J., Kazemzadeh-Atoufi, M., Ghoreishi, S., Blythe, S. A. and Garcia, H. G. (2020). Quantitative dissection of transcription in development yields evidence for transcription factor-driven chromatin accessibility. Elife 9. PubMed ID: 33074101
Summary:
Thermodynamic models of gene regulation can predict transcriptional regulation in bacteria, but in eukaryotes chromatin accessibility and energy expenditure may call for a different framework. This study systematically tested the predictive power of models of DNA accessibility based on the Monod-Wyman-Changeux (MWC) model of allostery, which posits that chromatin fluctuates between accessible and inaccessible states. The regulatory dynamics of hunchback by the activator Bicoid and the pioneer-like transcription factor Zelda was dissected in living Drosophila embryos; no thermodynamic or non-equilibrium MWC model could recapitulate hunchback transcription. Therefore, a model was explored where DNA accessibility is not the result of thermal fluctuations but is catalyzed by Bicoid and Zelda, possibly through histone acetylation; this model did predict hunchback dynamics. Thus, this theory-experiment dialogue uncovered potential molecular mechanisms of transcriptional regulatory dynamics, a key step toward reaching a predictive understanding of developmental decision-making.

Friday, January 8th - Disease models

Lee, S. H., Gomes, S. M., Ghalayini, J., Iliadi, K. G. and Boulianne, G. L. (2020). Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Blockers rescue memory defects in Drosophila expressing Alzheimer's disease-related transgenes independently of the canonical Renin Angiotensin System. eNeuro. PubMed ID: 33060184
Summary:
Alzheimer's disease (AD) is a degenerative disorder that causes progressive memory and cognitive decline. Recently, studies have reported that inhibitors of the mammalian renin angiotensin system (RAS) result in a significant reduction in the incidence and progression of AD by unknown mechanisms. This study used a genetic and pharmacological approach to evaluate the beneficial effects of Angiotensin Converting Enzyme Inhibitors (ACE-Is) and Angiotensin Receptor Blockers (ARBs) in Drosophila expressing AD-related transgenes. Importantly, while ACE orthologs have been identified in Drosophila, other RAS components are not conserved. This study shows that captopril, an ACE-I, and losartan, an ARB, can suppress a rough eye phenotype and brain cell death in flies expressing a mutant human C99 (C-terminal fragment of APP) transgene. Captopril also significantly rescues memory defects in these flies. Similarly, both drugs reduce cell death in Drosophila expressing human Aβ42, and losartan significantly rescues memory deficits. However, neither drug affects production, accumulation or clearance of Aβ42. Importantly, neither drug rescued brain cell death in Drosophila expressing human Tau suggesting that RAS inhibitors specifically target the amyloid pathway. Of note, reduced cell death and a complete rescue of memory deficits were observed when a null mutation in Drosophila Acer was crossed into each transgenic line demonstrating that the target of captopril in Drosophila is Acer. Altogether, these studies demonstrate that captopril and losartan are able to modulate AD related phenotypes in the absence of the canonical RAS pathway and suggest that both drugs have additional targets that can be identified in Drosophila

Lee, S., Kim, S., Kang, H. Y., Lim, H. R., Kwon, Y., Jo, M., Jeon, Y. M., Kim, S. R., Kim, K., Ha, C. M., Lee, S. and Kim, H. J. (2020). The overexpression of TDP-43 in astrocytes causes neurodegeneration via a PTP1B-mediated inflammatory response. J Neuroinflammation 17(1): 299. PubMed ID: 33054766
Summary:
Cytoplasmic inclusions of transactive response DNA binding protein of 43 kDa (TDP-43; see Drosophila TBPH) in neurons and astrocytes are a feature of some neurodegenerative diseases, such as frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). However, the role of TDP-43 in astrocyte pathology remains largely unknown. To investigate whether TDP-43 overexpression in primary astrocytes could induce inflammation, primary astrocytes were transfected with plasmids encoding Gfp or TDP-43-Gfp. The inflammatory response and upregulation of PTP1B in transfected cells were examined using quantitative RT-PCR and immunoblot analysis. Neurotoxicity was analysed in a transwell coculture system of primary cortical neurons with astrocytes and cultured neurons treated with astrocyte-conditioned medium (ACM). The lifespan was analyzed, climbing assays were performed and immunohistochemical data were analyzed in pan-glial TDP-43-expressing flies in the presence or absence of a Ptp61f RNAi transgene (the Drosophila homologue of PTP1B). PTP1B inhibition suppressed TDP-43-induced secretion of inflammatory cytokines (interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) in primary astrocytes. Using a neuron-astrocyte coculture system and astrocyte-conditioned media treatment, it was demonstrated that PTP1B inhibition attenuated neuronal death and mitochondrial dysfunction caused by overexpression of TDP-43 in astrocytes. In addition, in Drosophila, neuromuscular junction (NMJ) defects, a shortened lifespan, inflammation and climbing defects caused by pan-glial overexpression of TDP-43 were significantly rescued by downregulation of ptp61f. These results indicate that PTP1B inhibition mitigates the neuronal toxicity caused by TDP-43-induced inflammation in mammalian astrocytes and Drosophila glial cells.

Han, M. H., Kwon, M. J., Ko, B. S., Hyeon, D. Y., Lee, D., Kim, H. J., Hwang, D. and Lee, S. B. (2020). NF-kappaB disinhibition contributes to dendrite defects in fly models of neurodegenerative diseases. J Cell Biol 219(12). PubMed ID: 33090185
Summary:
Dendrite pathology is frequently observed in various neurodegenerative diseases (NDs). Although previous studies identified several pathogenic mediators of dendrite defects that act through loss of function in NDs, the underlying pathogenic mechanisms remain largely unexplored. A search for additional pathogenic contributors to dendrite defects in NDs identified Relish/NF-kappaB as a novel gain-of-toxicity-based mediator of dendrite defects in animal models for polyglutamine (polyQ) diseases and amyotrophic lateral sclerosis (ALS). In a Drosophila model for polyQ diseases, polyQ-induced dendrite defects require Dredd/Caspase-8-mediated endoproteolytic cleavage of Relish to generate the N-terminal fragment, Rel68, and subsequent Charon-mediated nuclear localization of Rel68. Rel68 alone induced neuronal toxicity causing dendrite and behavioral defects, and two novel transcriptional targets, Tup and Pros, were identified that mediate Rel68-induced neuronal toxicity. Finally, it was shown that Rel68-induced toxicity also contributes to dendrite and behavioral defects in a Drosophila model for ALS. Collectively, these data propose disinhibition of latent toxicity of Relish/NF-κB as a novel pathogenic mechanism underlying dendrite pathology in NDs.

Delfino, L., Mason, R. P., Kyriacou, C. P., Giorgini, F. and Rosato, E. (2020). Rab8 Promotes Mutant HTT Aggregation, Reduces Neurodegeneration, and Ameliorates Behavioural Alterations in a Drosophila Model of Huntington's Disease. J Huntingtons Dis 9(3): 253-263. PubMed ID: 33044189
Summary:
Altered cellular vesicle trafficking has been linked to the pathogenesis of Huntington's disease (HD), a fatal, inherited neurodegenerative disorder caused by mutation of the huntingtin (HTT) protein. The Rab GTPase family of proteins plays a key role in regulation of vesicle trafficking. This study investigated whether Rab8 (see Drosophila Rab8), which regulates post-Golgi vesicle trafficking, is able to improve HD-relevant phenotypes in a well-characterised model. Rab8 was overexpressed in a Drosophila model of HD, testing cellular, behavioural, and molecular phenotypes. Rab8 overexpression ameliorated several disease-related phenotypes in fruit flies expressing a mutant HTT (see Drosophila Huntingtin) fragment throughout the nervous system, including neurodegeneration of photoreceptor neurons, reduced eclosion of the adult fly from the pupal case and shortened lifespan. Rab8 overexpression also normalised aberrant circadian locomotor behaviour in flies expressing mutant HTT in a specific population of neurons that regulate the circadian clock. Intriguingly, expression of Rab8 increased the accumulation of SDS-insoluble aggregated species of mutant HTT. Collectively, these findings demonstrate that increased Rab8 levels protect against mutant HTT toxicity and potentiate its aggregation, likely reducing the accumulation of downstream toxic soluble species.

Mollasalehi, N., Francois-Moutal, L., Scott, D. D., Tello, J. A., Williams, H., Mahoney, B., Carlson, J. M., Dong, Y., Li, X., Miranda, V. G., Gokhale, V., Wang, W., Barmada, S. J. and Khanna, M. (2020). An Allosteric Modulator of RNA Binding Targeting the N-Terminal Domain of TDP-43 Yields Neuroprotective Properties. ACS Chem Biol 15(11): 2854-2859. PubMed ID: 33044808
Summary:
This study targeted the N-terminal domain (NTD) of transactive response (TAR) DNA binding protein (TDP-43), which is implicated in several neurodegenerative diseases. In silico docking of 50K compounds to the NTD domain of TDP-43 identified a small molecule (nTRD22) that is bound to the N-terminal domain. Interestingly, nTRD22 caused allosteric modulation of the RNA binding domain (RRM) of TDP-43, resulting in decreased binding to RNA in vitro. Moreover, incubation of primary motor neurons with nTRD22 induced a reduction of TDP-43 protein levels, similar to TDP-43 RNA binding-deficient mutants and supporting a disruption of TDP-43 binding to RNA. Finally, nTRD22 mitigated motor impairment in a Drosophila model of amyotrophic lateral sclerosis. These findings provide an exciting way of allosteric modulation of the RNA-binding region of TDP-43 through the N-terminal domain.

Balasov, M., Akhmetova, K. and Chesnokov, I. (2020). Humanized Drosophila Model of the Meier-Gorlin Syndrome Reveals Conserved and Divergent Features of the Orc6 Protein. Genetics. PubMed ID: 33037049. Journal
Summary:
Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears and skeletal abnormalities. Patients with MGS often carry mutations in genes encoding the subunits of the Origin Recognition Complex (ORC), components of the pre-replicative complex (pre-RC) and replication machinery. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. A mutation in the conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. Recently, a new mutation in Orc6 was also identified however, it is localized in the N-terminal domain of the protein. In order to study the functions of Orc6, the human gene was used to rescue the orc6 deletion in Drosophila. Using this "humanized" Orc6-based Drosophila model of the Meier-Gorlin syndrome it was discovered that unlike the previous Y225S MGS mutation in Orc6, the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. These studies revealed the importance of evolutionarily conserved and variable domains of Orc6 protein and allowed the studies of human protein functions and the analysis of the critical amino acids in live animal heterologous system as well as provided novel insights into the mechanisms underlying MGS pathology.

Thursday, January 7th - Signal Transduction

He, Q., Du, J., Wei, L. and Zhao, Z. (2020). AKH-FOXO pathway regulates starvation-induced sleep loss through remodeling of the small ventral lateral neuron dorsal projections. PLoS Genet 16(10): e1009181. PubMed ID: 33104699
Summary:
Starvation caused by adverse feeding stresses or food shortages has been reported to result in sleep loss in animals. However, how the starvation signal interacts with the central nervous system is still unknown. In this study, the adipokinetic hormone (AKH)-Fork head Box-O (FOXO) pathway is shown to respond to energy change and adjust the sleep of Drosophila through remodeling of the s-LNv (small ventral lateral neurons) dorsal projections. The results show that starvation prevents flies from going to sleep after the first light-dark transition. The LNvs are required for starvation-induced sleep loss through extension of the pigment dispersing factor (PDF)-containing s-LNv dorsal projections. Further studies reveal that loss of AKH or AKHR (akh receptor) function blocks starvation-induced extension of s-LNv dorsal projections and rescues sleep suppression during food deprivation. FOXO, which has been reported to regulate synapse plasticity of neurons, acts as starvation response factor downstream of AKH, and down regulation of FOXO level considerably alleviates the influence of starvation on s-LNv dorsal projections and sleep. Taking together, these results outline the transduction pathways between starvation signal and sleep, and reveal a novel functional site for sleep regulation.

Ge, M., Zhang, T., Zhang, M. and Cheng, L. (2020). Ran participates in deltamethrin stress through regulating the nuclear import of Nrf2. Gene: 145213. PubMed ID: 33069802
Summary:
The small GTPase Ran has a variety of biological functions, one of the most prominent of which is to regulate nucleocytoplasmic transport. In a previous study, it was suggested that Ran is involved in the deltamethrin (DM) stress. In addition, Keap1-Nrf2-ARE pathway was also confirmed to be associated with DM stress. This study reports that under DM stress, interfering Ran or nuclear transport factor Ntf2 by RNAi could suppress the nuclear import of nuclear transcription factor Nrf2 which then down-regulates the expressions of detoxification enzyme genes (Cyp4d20, Cyp4ae1, GstD5, Sod3, etc.), ultimately resulting in a significant apoptosis of Drosophila Kc cells. In contrast, after overexpressing Ran in Kc cells, Nrf2 has a higher concentration in the nucleus, and the expressions of detoxification enzyme genes are up-regulated, while the DM-induced apoptosis is significantly lower than that of the control group. Additionally, it was preliminary found that silencing Ntf2 or Ran could prevent the nuclear import of transcription factor Dif under DM stress, subsequently decreased expressions of antimicrobial peptide genes (Drsl1). In summary, the data mainly indicates that Ran may participate in DM stress through regulating the nuclear import of Nrf2, which could help to study the mechanism of deltamethrin resistance.

Hatori, R. and Kornberg, T. B. (2020). Hedgehog produced by the Drosophila wing imaginal disc induces distinct responses in three target tissues. Development 147(22). PubMed ID: 33028613
Summary:
Hedgehog (Hh) is an evolutionarily conserved signaling protein that has essential roles in animal development and homeostasis. This study investigated Hh signaling in the region of the Drosophila wing imaginal disc that produces Hh and is near the tracheal air sac primordium (ASP) and myoblasts. Hh distributes in concentration gradients in the anterior compartment of the wing disc, ASP and myoblasts, and activates genes in each tissue. Some targets of Hh signal transduction are common to the disc, ASP and myoblasts, whereas others are tissue-specific. Signaling in the three tissues is cytoneme-mediated and cytoneme-dependent. Some ASP cells project cytonemes that receive both Hh and Branchless (Bnl), and some targets regulated by Hh signaling in the ASP are also dependent on Bnl signal transduction. It is concluded that the single source of Hh in the wing disc regulates cell type-specific responses in three discreet target tissues.

Ghosh, A. C., Tattikota, S. G., Liu, Y., Comjean, A., Hu, Y., Barrera, V., Ho Sui, S. J. and Perrimon, N. (2020). Drosophila PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity. Elife 9. PubMed ID: 33107824
Summary:
PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine and endocrine mechanisms. This study investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). Genetic approaches and single-nuclei sequencing were combined to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. This study found that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. These findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells

Little, J. C., Garcia-Garcia, E., Sul, A. and Kalderon, D. (2020). Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing. Elife 9. PubMed ID: 33084577
Summary:
Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. This study used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.

Mesrouze, Y., Aguilar, G., Bokhovchuk, F., Martin, T., Delaunay, C., Villard, F., Meyerhofer, M., Zimmermann, C., Fontana, P., Wille, R., Vorherr, T., Erdmann, D., Furet, P., Scheufler, C., Schmelzle, T., Affolter, M. and Chene, P. (2020). A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors. Sci Rep 10(1): 17442. PubMed ID: 33060790
Summary:
The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and in this study on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, these studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, it is proposed that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

Wednesday, January 6th - Adult physiology

McMullen, J. G., Peters-Schulze, G., Cai, J., Patterson, A. D. and Douglas, A. E. (2020). How gut microbiome interactions affect nutritional traits of Drosophila melanogaster. J Exp Biol 223(Pt 19). PubMed ID: 33051361
Summary:
Most research on the impact of the gut microbiome on animal nutrition is designed to identify the effects of single microbial taxa and single metabolites of microbial origin, without considering the potentially complex network of interactions among co-occurring microorganisms. This study investigated how different microbial associations and their fermentation products affect host nutrition, using Drosophila melanogaster colonized with three gut microorganisms (the bacteria Acetobacter fabarum and Lactobacillus brevis, and the yeast Hanseniaspora uvarum) in all seven possible combinations. Some microbial effects on host traits could be attributed to single taxa (e.g. yeast-mediated reduction of insect development time), while other effects were sex specific and driven by among-microbe interactions (e.g. male lipid content determined by interactions between the yeast and both bacteria). Parallel analysis of nutritional indices of microbe-free flies administered different microbial fermentation products (acetic acid, acetoin, ethanol and lactic acid) revealed a single consistent effect: that the lipid content of both male and female flies is reduced by acetic acid. This effect was recapitulated in male flies colonized with both yeast and A. fabarum, but not for any microbial treatment in females or males with other microbial complements. These data suggest that the effect of microbial fermentation products on host nutritional status is strongly context dependent, with respect to both the combination of associated microorganisms and host sex. Taken together, these findings demonstrate that among-microbe interactions can play a critically important role in determining the physiological outcome of host-microbiome interactions in Drosophila and, likely, in other animal hosts.

Carneiro Dutra, H. L., Deehan, M. A. and Frydman, H. (2020). Wolbachia and Sirtuin-4 interaction is associated with alterations in host glucose metabolism and bacterial titer. PLoS Pathog 16(10): e1008996. PubMed ID: 33048997
Summary:
Wolbachia is an intracellular bacterial symbiont of arthropods notorious for inducing many reproductive manipulations that foster its dissemination. Sirtuins (SIRTs) are a family of NAD+-dependent post-translational regulatory enzymes known to affect many of the same processes altered by Wolbachia, including aging and metabolism, among others. Despite a clear overlap in control of host-derived pathways and physiology, no work has demonstrated a link between these two regulators. This study used genetically tractable Drosophila melanogaster to explore the role of sirtuins in shaping signaling pathways in the context of a host-symbiont model. By using transcriptional profiling and metabolic assays in the context of genetic knockouts/over-expressions, the effect of several Wolbachia strains on host sirtuin expression was examined across distinct tissues and timepoints. The downstream effects of the sirtuin x Wolbachia interaction were examined on host glucose metabolism, and in turn, how it impacted Wolbachia titer. The results indicate that the presence of Wolbachia is associated with (1) reduced sirt-4 expression in a strain-specific manner, and (2) alterations in host Glutamate dehydrogenase expression and ATP levels, key components of glucose metabolism. High glucose levels were detected in Wolbachia-infected flies, which further increased when sirt-4 was over-expressed. However, under sirt-4 knockout, flies displayed a hypoglycemic state not rescued to normal levels in the presence of Wolbachia. Finally, whole body sirt-4 over-expression resulted in reduced Wolbachia ovarian titer. These results expand knowledge of Wolbachia-host associations in the context of a yet unexplored class of host post-translational regulatory enzymes with implications for conserved host signaling pathways and bacterial titer, factors known to impact host biology and the symbiont's ability to spread through populations.

Dai, Z., Li, D., Du, X., Ge, Y., Hursh, D. A. and Bi, X. (2020). Drosophila Caliban preserves intestinal homeostasis and lifespan through regulating mitochondrial dynamics and redox state in enterocytes. PLoS Genet 16(10): e1009140. PubMed ID: 33057338
Summary:
Precise regulation of stem cell activity is crucial for tissue homeostasis. In Drosophila, intestinal stem cells (ISCs) maintain the midgut epithelium and respond to oxidative challenges. However, the connection between intestinal homeostasis and redox signaling remains obscure. This study found that Caliban (Clbn), a component of the ribosome quality control complex (RQC), functions as a regulator of mitochondrial dynamics in enterocytes (ECs) and is required for intestinal homeostasis. The clbn knock-out flies have a shortened lifespan and lose the intestinal homeostasis. Clbn is highly expressed and localizes to the outer membrane of mitochondria in ECs. Mechanically, Clbn mediates mitochondrial dynamics in ECs and removal of clbn leads to mitochondrial fragmentation, accumulation of reactive oxygen species, ECs damage, activation of JNK and JAK-STAT signaling pathways. Moreover, multiple mitochondria-related genes are differentially expressed between wild-type and clbn mutated flies by a whole-genome transcriptional profiling. Furthermore, loss of clbn promotes tumor growth in gut generated by activated Ras in intestinal progenitor cells. These findings reveal an EC-specific function of Clbn in regulating mitochondrial dynamics, and provide new insight into the functional link among mitochondrial redox modulation, tissue homeostasis and longevity.

Morgante, F., Huang, W., Sorensen, P., Maltecca, C. and Mackay, T. F. C. (2020). Leveraging multiple layers of data to predict Drosophila complex traits. G3 (Bethesda). PubMed ID: 33106232
Summary:
The ability to accurately predict complex trait phenotypes from genetic and genomic data is critical for the implementation of personalized medicine and precision agriculture; however, prediction accuracy for most complex traits is currently low. This study used data on whole genome sequences, deep RNA sequencing, and high quality phenotypes for three quantitative traits in the ~200 inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) to compare the prediction accuracies of gene expression and genotypes for three complex traits. Expression levels provided higher prediction accuracy than genotypes for starvation resistance, similar prediction accuracy for chill coma recovery (null for both models and sexes), and lower prediction accuracy for startle response . Models including both genotype and expression levels did not outperform the best single component model. However, accuracy increased considerably for all the three traits when gene ontology (GO) category was included as an additional layer of information for both genomic variants and transcripts. Strongly predictive GO terms were found for each of the three traits, some of which had a clear plausible biological interpretation. For example, for starvation resistance in females, GO:0033500 and GO:0032870, have been implicated in carbohydrate homeostasis and cellular response to hormone stimulus (including the insulin receptor signaling pathway), respectively. In summary, this study shows that integrating different sources of information improved prediction accuracy and helped elucidate the genetic architecture of three Drosophila complex phenotypes.

McCracken, A. W., Buckle, E. and Simons, M. J. P. (2020). The relationship between longevity and diet is genotype dependent and sensitive to desiccation in Drosophila melanogaster. J Exp Biol. PubMed ID: 33109715
Summary:
Dietary restriction (DR) is a key focus in ageing research. Specific conditions and genotypes were recently found to negate lifespan extension by DR, questioning its universal relevance. However, the concept of dietary reaction norms explains why DR's effects might be obscured in some situations. This study tested the importance of dietary reaction norms by measuring longevity and fecundity on five diets in five genotypes, with and without water supplementation in female Drosophila melanogaster (N>25,000). Substantial genetic variation was found in the response of lifespan to diet. Flies supplemented with water rescued putative desiccation stress at the richest diets, suggesting water availability can be an experimental confound. Fecundity declined at these richest diets, but was unaffected by water, and this reduction is thus most likely caused by nutritional toxicity. These results demonstrate empirically that a range of diets need to be considered to conclude an absence of the DR longevity effect.

Duhart, J. M., Herrero, A., de la Cruz, G., Ispizua, J. I., Pirez, N. and Ceriani, M. F. (2020). Circadian structural plasticity drives remodeling of E cell output. Curr Biol. PubMed ID: 33065014
Summary:
The Drosophila circadian network dictates the temporal organization of locomotor activity; under light-dark (LD) conditions, flies display a robust bimodal pattern. PDF-positive small ventral lateral neurons (sLNv) have been linked to the generation of the morning activity peak (the "M cells"), whereas the CRY-positive dorsal lateral neurons (LNds) and the PDF-negative sLNv are necessary for the evening activity peak (the "E cells"). While each group directly controls locomotor output pathways, an interplay between them along with a third dorsal cluster (the DN1ps) is necessary for the correct timing of each peak and for adjusting behavior to changes in the environment. M cells set the phase of roughly half of the circadian neurons (including the E cells) through PDF. This study shows the existence of synaptic input provided by the evening oscillator onto the M cells. Both structural and functional approaches revealed that E-to-M cell connectivity changes across the day, with higher excitatory input taking place before the day-to-night transition. Two different neurotransmitters, acetylcholine and glutamate, released by E cells were identified that are relevant for robust circadian output. Indeed, this study shows that acetylcholine is responsible for the excitatory input from E cells to M cells, which show preferential responsiveness to acetylcholine during the evening. These findings provide evidence of an excitatory feedback between circadian clusters and unveil an important plastic remodeling of the E cells' synaptic connections.

Tuesday January 5th - Adult neural development and function

Gao, Y., Zhu, C., Li, K., Cheng, X., Du, Y., Yang, D., Fan, X., Gaur, U. and Yang, M. (2020). Comparative proteomics analysis of dietary restriction in Drosophila. PLoS One 15(10): e0240596. PubMed ID: 33064752
Summary:
To explore the underlying mechanism of dietary restriction (DR) induced lifespan extension in fruit flies at protein level, proteome sequencing was performed in Drosophila at day 7 (young) and day 42 (old) under DR and ad libitum (AL) conditions. A total of 18629 unique peptides were identified in Uniprot, corresponding to 3,662 proteins. Among them, 383 and 409 differentially expressed proteins (DEPs) were identified from comparison between DR vs AL at day 7 and 42, respectively. Bioinformatics analysis revealed that membrane-related processes, post-transcriptional processes, spliceosome and reproduction related processes, were highlighted significantly. In addition, expression of proteins involved in pathways such as spliceosomes, oxidative phosphorylation, lysosomes, ubiquitination, and riboflavin metabolism was relatively higher during DR. A relatively large number of DEPs were found to participate in longevity and age-related disease pathways. 20 proteins were identified that were consistently regulated during DR and some of which are known to be involved in ageing, such as mTORC1, antioxidant, DNA damage repair and autophagy. In the integration analysis, 15 genes were found that were stably regulated by DR at both transcriptional as well as translational levels. These results provided a useful dataset for further investigations on the mechanism of DR and aging.

Farnworth, M. S., Eckermann, K. N. and Bucher, G. (2020). Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: A fly-beetle insight. PLoS Biol 18(10): e3000881. PubMed ID: 33104689
Summary:
Animal behavior is guided by the brain. Therefore, adaptations of brain structure and function are essential for animal survival, and each species differs in such adaptations. The brain of one individual may even differ between life stages, for instance, as adaptation to the divergent needs of larval and adult life of holometabolous insects. All such differences emerge during development, but the cellular mechanisms behind the diversification of brains between taxa and life stages remain enigmatic. This study investigated holometabolous insects in which larvae differ dramatically from the adult in both behavior and morphology. As a consequence, the central complex, mainly responsible for spatial orientation, is conserved between species at the adult stage but differs between larvae and adults of one species as well as between larvae of different taxa. This study used genome editing and established transgenic lines to visualize cells expressing the conserved transcription factor Retinal homeobox, thereby marking homologous genetic neural lineages in both the fly Drosophila melanogaster and the beetle Tribolium castaneum. This approach allowed comparison of the development of homologous neural cells between taxa from embryo to the adult. Complex heterochronic changes were found including shifts of developmental events between embryonic and pupal stages. Further, the first example of sequence heterochrony in brain development was found, where certain developmental steps changed their position within the ontogenetic progression. Through this sequence heterochrony, an immature developmental stage of the central complex gains functionality in Tribolium larvae.

Sears, J. C. and Broadie, K. (2020). FMRP-PKA activity negative feedback regulates RNA binding-dependent fibrillation in brain learning and memory circuitry. Cell Rep 33(2): 108266. PubMed ID: 33053340
Summary:
Fragile X mental retardation protein (FMRP) promotes cyclic AMP (cAMP) signaling. Using an in vivo protein kinase A activity sensor (PKA-SPARK), this study found that Drosophila FMRP (dFMRP) and human FMRP (hFMRP) enhance PKA activity in a central brain learning and memory center. Increasing neuronal PKA activity suppresses FMRP in Kenyon cells, demonstrating an FMRP-PKA negative feedback loop. A patient-derived R140Q FMRP point mutation mislocalizes PKA-SPARK activity, whereas deletion of the RNA-binding arginine-glycine-glycine (RGG) box (hFMRP-ΔRGG) produces fibrillar PKA-SPARK assemblies colocalizing with ribonucleoprotein (RNP) and aggregation (thioflavin T) markers, demonstrating fibrillar partitioning of cytosolic protein aggregates. hFMRP-ΔRGG reduces dFMRP levels, indicating RGG-independent regulation. Short-term hFMRP-ΔRGG induction produces activated PKA-SPARK puncta, whereas long induction drives fibrillar assembly. Elevated temperature disassociates hFMRP-ΔRGG aggregates and blocks activated PKA-SPARK localization. These results suggest that FMRP regulates compartmentalized signaling via complex assembly, directing PKA activity localization, with FMRP RGG box RNA binding restricting separation via low-complexity interactions.

Flyer-Adams, J. G., Rivera-Rodriguez, E. J., Yu, J., Mardovin, J. D., Reed, M. L. and Griffith, L. C. (2020). Regulation of olfactory associative memory by the circadian clock output signal Pigment-dispersing factor (PDF). J Neurosci 40(47): 9066-9077. PubMed ID: 33106351
Summary:
Dissociation between the output of the circadian clock and external environmental cues is a major cause of human cognitive dysfunction. While the effects of ablation of the molecular clock on memory have been studied in many systems, little has been done to test the role of specific clock circuit output signals. To address this gap, this study examined the effects of mutations of Pigment-dispersing factor (Pdf) and its receptor, Pdfr, on associative memory in male and female Drosophila. Loss of PDF signaling significantly decreases the ability to form associative memory. Appetitive short-term memory (STM), which in wild-type (WT) is time-of-day (TOD) independent, is decreased across the day by mutation of Pdf or Pdfr, but more substantially in the morning than in the evening. This defect is because of PDFR expression in adult neurons outside the core clock circuit and the mushroom body (MB) Kenyon cells (KCs). The acquisition of a TOD difference in mutants implies the existence of multiple oscillators that act to normalize memory formation across the day for appetitive processes. Interestingly, aversive STM requires PDF but not PDFR, suggesting that there are valence-specific pathways downstream of PDF that regulate memory formation. These data argue that the circadian clock uses circuit-specific and molecularly diverse output pathways to enhance the ability of animals to optimize responses to changing conditions.

Hu, Y., Wang, C., Yang, L., Pan, G., Liu, H., Yu, G. and Ye, B. (2020). A neural basis for categorizing sensory stimuli to enhance decision accuracy. Curr Biol. PubMed ID: 33065003
Summary:
Sensory stimuli with graded intensities often lead to yes-or-no decisions on whether to respond to the stimuli. How this graded-to-binary conversion is implemented in the central nervous system (CNS) remains poorly understood. This study shows that graded encodings of noxious stimuli are categorized in a decision-associated CNS region in the ventral cord of Drosophila larvae, and then decoded by a group of peptidergic neurons for executing binary escape decisions. GABAergic inhibition gates weak nociceptive encodings from being decoded, whereas escalated amplification through the recruitment of second-order neurons boosts nociceptive encodings at intermediate intensities. These two modulations increase the detection accuracy by reducing responses to negligible stimuli whereas enhancing responses to intense stimuli. These findings thus unravel a circuit mechanism that underlies accurate detection of harmful stimuli.

Hampel, S., Eichler, K., Yamada, D., Bock, D. D., Kamikouchi, A. and Seeds, A. M. (2020). Distinct subpopulations of mechanosensory chordotonal organ neurons elicit grooming of the fruit fly antennae. Elife 9. PubMed ID: 33103999
Summary:
Diverse mechanosensory neurons detect different mechanical forces that can impact animal behavior. Yet understanding of the anatomical and physiological diversity of these neurons and the behaviors that they influence is limited. It was previously discovered that grooming of the Drosophila melanogaster antennae is elicited by an antennal mechanosensory chordotonal organ, the Johnston's organ (JO). This paper describes anatomically and physiologically distinct JO mechanosensory neuron subpopulations that each elicit antennal grooming. The subpopulations project to different, discrete zones in the brain and differ in their responses to mechanical stimulation of the antennae. Although activation of each subpopulation elicits antennal grooming, distinct subpopulations also elicit the additional behaviors of wing flapping or backward locomotion. These results provide a comprehensive description of the diversity of mechanosensory neurons in the JO, and reveal that distinct JO subpopulations can elicit both common and distinct behavioral responses.

Monday, January 4th - Evolution

Vlachakis, D., Papageorgiou, L., Papadaki, A., Georga, M., Kossida, S. and Eliopoulos, E. (2020). An updated evolutionary study of the Notch family reveals a new ancient origin and novel invariable motifs as potential pharmacological targets. PeerJ 8: e10334. PubMed ID: 33194454
Summary:
Notch family proteins play a key role in a variety of developmental processes by controlling cell fate decisions and operating in a great number of biological processes in several organ systems, such as hematopoiesis, somatogenesis, vasculogenesis, neurogenesis and homeostasis. The Notch signaling pathway is crucial for the majority of developmental programs and regulates multiple pathogenic processes. Notch family receptors' activation has been largely related to its multiple effects in sustaining oncogenesis. The Notch signaling pathway constitutes an ancient and conserved mechanism for cell to cell communication. Much of what is known about Notch family proteins function comes from studies done in Caenorhabditis elegans and Drosophila melanogaster. Although, human Notch homologs had also been identified, the molecular mechanisms which modulate the Notch signaling pathway remained substantially unknown. In this study, an updated evolutionary analysis of the Notch family members among 603 different organisms of all kingdoms, from bacteria to humans, was performed in order to discover key regions that have been conserved throughout evolution and play a major role in the Notch signaling pathway. The major goal of this study is the presentation of a novel updated phylogenetic tree for the Notch family as a reliable phylogeny "map", in order to correlate information of the closely related members and identify new possible pharmacological targets that can be used in pathogenic cases, including cancer.

Ahmed-Braimah, Y. H., Wolfner, M. F. and Clark, A. G. (2020). Differences in post-mating transcriptional responses between conspecific and heterospecific matings in Drosophila. Mol Biol Evol. PubMed ID: 33035303
Summary:
In many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins, and are critical for fertilization success. Unfortunately, understanding of the molecular interplay between female and male reproductive proteins remains inadequate. This study analyzed the post-mating response in a Drosophila species that has evolved strong gametic incompatibility with its sister species; D. novamexicana females produce only ~1% fertilized eggs in crosses with D. americana males, compared to ~98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study short-read RNA sequencing was used to examine the evolutionary dynamics of female reproductive genes and the post-mating transcriptome response in crosses within and between species. First, it was found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, post-mating responses in con- and heterospecific matings are largely congruent, but heterospecific matings induce expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. Several genes in the JAK/STAT signaling pathway were identified that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, it was also detected in the female head and ovaries. These results show that the female's post-mating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females.

Agren, J. A., Munasinghe, M. and Clark, A. G. (2020). Mitochondrial-Y chromosome epistasis in Drosophila melanogaster. Proc Biol Sci 287(1937): 20200469. PubMed ID: 33081607
Summary:
The coordination between mitochondrial and nuclear genes is crucial to eukaryotic organisms. Predicting the nature of these epistatic interactions can be difficult because of the transmission asymmetry of the genes involved. While autosomes and X-linked genes are transmitted through both sexes, genes on the Y chromosome and in the mitochondrial genome are uniparentally transmitted through males and females, respectively. This study generated 36 otherwise isogenic Drosophila melanogaster strains differing only in the geographical origin of their mitochondrial genome and Y chromosome, to experimentally examine the effects of the uniparentally inherited parts of the genome, as well as their interaction, in males. Longevity and gene expression was assayed through RNA-sequencing. An important role was detected for both mitochondrial and Y-linked genes, as well as extensive mitochondrial-Y chromosome epistasis. In particular, genes involved in male reproduction appear to be especially sensitive to such interactions, and variation on the Y chromosome is associated with differences in longevity. Despite these interactions, no evidence was found that the mitochondrial genome and Y chromosome are co-adapted within a geographical region. Overall, this study demonstrates a key role for the uniparentally inherited parts of the genome for male biology, but also that mito-nuclear interactions are complex and not easily predicted from simple transmission asymmetries.

White, L. A., Siva-Jothy, J. A., Craft, M. E. and Vale, P. F. (2020). Genotype and sex-based host variation in behaviour and susceptibility drives population disease dynamics. Proc Biol Sci 287(1938): 20201653. PubMed ID: 33171094
Summary:
Host heterogeneity in pathogen transmission is widespread and presents a major hurdle to predicting and minimizing disease outbreaks. Using Drosophila melanogaster infected with Drosophila C virus as a model system, this study integrated experimental measurements of social aggregation, virus shedding, and disease-induced mortality from different genetic lines and sexes into a disease modelling framework. The experimentally measured host heterogeneity produced substantial differences in simulated disease outbreaks, providing evidence for genetic and sex-specific effects on disease dynamics at a population level. While this was true for homogeneous populations of single sex/genetic line, the genetic background or sex of the index case did not alter outbreak dynamics in simulated, heterogeneous populations. Finally, to explore the relative effects of social aggregation, viral shedding and mortality, simulations were compared where these traits were allowed to vary, as measured experimentally, to simulations where variation in these traits was constrained to the population mean. In this context, variation in infectiousness, followed by social aggregation, was the most influential component of transmission. Overall, this study shows that host heterogeneity in three host traits dramatically affects population-level transmission, but the relative impact of this variation depends on both the susceptible population diversity and the distribution of population-level variation.

Ward, H. and Moehring, A. J. (2020). Genes underlying species differences in CHC production between Drosophila melanogaster and D. simulans. Genome. PubMed ID: 33211537
Summary:
Surface chemical compounds are key components of survival and reproduction in many species. Cuticular hydrocarbons (CHCs) are chemical compounds produced by all insects that are used for both desiccation resistance and chemical communication, including communication related to mating. In the species pair of Drosophila melanogaster and D. simulans, female CHCs stimulate conspecific males to mate and repel heterospecific males. While CHCs are a critical contributor to both reproductive success within a species and isolation between species, few genes underlying species variation in CHC profiles are known. This study used genetic mapping of the 3rd chromosome to test a suite of candidate genes for interspecies variation in CHCs. Candidate gene CG5946 was found to be involved in species differences in the production of 7,11-heptacosadiene and 7-tricosene between D. melanogaster and D. simulans. This is therefore a new candidate locus contributing to species-specific variation in the CHC profile. In the process of mapping genes for CHCs, 29 candidate genes for the reduced survival or inviability of interspecies hybrids were identified.

Mallard, F., Nolte, V. and Schlotterer, C. (2020). The evolution of phenotypic plasticity in response to temperature stress. Genome Biol Evol. PubMed ID: 33022043
Summary:
Phenotypic plasticity is the ability of a single genotype to produce different phenotypes in response to environmental variation. The importance of phenotypic plasticity in natural populations and its contribution to phenotypic evolution during rapid environmental change is widely debated. This study shows that thermal plasticity of gene expression in natural populations is a key component of its adaptation: evolution to novel thermal environments increases ancestral plasticity rather than mean genetic expression. The evolution of plasticity in gene expression was determined by conducting laboratory natural selection on a Drosophila simulans population in hot and cold environments. After more than 60 generations in the hot environment, 325 genes evolved a change in plasticity relative to the natural ancestral population. Plasticity increased in 75% of these genes, which were strongly enriched for several well-defined functional categories (e.g. chitin metabolism, glycolysis and oxidative phosphorylation). Furthermore, this study showed that plasticity in gene expression of populations exposed to different temperatures is rather similar across species. It is concluded that most of the ancestral plasticity can evolve further in more extreme environments.

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