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Current papers in developmental biology and gene function


April 30th, 2019 - Synapse and Vesicles

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Politano, S. F., Salemme, R. R., Ashley, J., Lopez-Rivera, J. A., Bakula, T. A., Puhalla, K. A., Quinn, J. P., Juszczak, M. J., Phillip, L. K., Carrillo, R. A. and Vanderzalm, P. J. (2019). Tao negatively regulates BMP signaling during neuromuscular junction development in Drosophila. Dev Neurobiol. PubMed ID: 31002474
The coordinated growth and development of synapses is critical for all aspects of neural circuit function and mutations that disrupt these processes can result in various neurological defects. Several anterograde and retrograde signaling pathways, including the canonical Bone Morphogenic Protein (BMP) pathway, regulate synaptic development in vertebrates and invertebrates. At the Drosophila larval neuromuscular junction (NMJ), the retrograde BMP pathway is part of the machinery that controls NMJ expansion concurrent with larval growth. This study sought to determine whether the conserved Hippo pathway, critical for proportional growth in other tissues, also functions in NMJ development. Neuronal loss of the serine-threonine protein kinase Tao, a regulator of the Hippo signaling pathway, results in supernumerary boutons, each of which contain a normal number of active zones. Tao is also required for proper synaptic function, as reduction of Tao results in NMJs with decreased evoked excitatory junctional potentials. Surprisingly, Tao function in NMJ growth is independent of the Hippo pathway. Instead, the experiments suggest that Tao negatively regulates BMP signaling as reduction of Tao leads to an increase in pMad levels in motor neuron nuclei and an increase in BMP target gene expression. Taken together, these results support a role for Tao as a novel inhibitor of BMP signaling in motor neurons during synaptic development and function.
Vernon, S. W., Goodchild, J. and Baines, R. A. (2019). A poly-glutamine region in the Drosophila VAChT dictates fill-level of cholinergic synaptic vesicles. eNeuro 6(1). PubMed ID: 30847389
While the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles (SVs), accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. This study extend the role of the VAChT to include regulating the transmitter content of SVs. Manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, it was found that reduction of the polyQ region, by one glutamine residue (13Q to 12Q), results in a significant increase in both amplitude and frequency of spontaneous cholinergic miniature EPSCs (mEPSCs) recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine (13Q to 14Q) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked SV release. Finally, a complete deletion of the polyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure SVs are filled to physiologically-appropriate levels remain unknown. This study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission.
Scholz, N., Ehmann, N., Sachidanandan, D., Imig, C., Cooper, B. H., Jahn, O., Reim, K., Brose, N., Meyer, J., Lamberty, M., Altrichter, S., Bormann, A., Hallermann, S., Pauli, M., Heckmann, M., Stigloher, C., Langenhan, T. and Kittel, R. J. (2019). Complexin cooperates with Bruchpilot to tether synaptic vesicles to the active zone cytomatrix. J Cell Biol 218(3): 1011-1026. PubMed ID: 30782781
Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. This study identifies the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.
Tao, J., Bulgari, D., Berkhoudt, D. A., Calderon, M. J., Watkins, S. C., Fonseca, H., Sabeva, N., Deitcher, D. L. and Levitan, E. S. (2019). Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission. J Cell Sci. PubMed ID: 30837287
Many neurons influence their targets through co-release of neuropeptides and small molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release, as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both STED super-resolution and transmission electron microscopy. Ptp4E also controls presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content.
Zhao, G., Oztan, A., Ye, Y. and Schwarz, T. L. (2019). Kinetochore proteins have a post-mitotic function in neurodevelopment. Dev Cell. PubMed ID: 30827899
The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. This study shows that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. It is concluded that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals.
Boda, A., Lorincz, P., Takats, S., Csizmadia, T., Toth, S., Kovacs, A. L. and Juhasz, G. (2019). Drosophila Arl8 is a general positive regulator of lysosomal fusion events. Biochim Biophys Acta Mol Cell Res 1866(4): 533-544. PubMed ID: 30590083
The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. This study shows that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, this study demonstrates that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor.

Monday, April 29th - Adult Neural Function

Zhou, M., Chen, N., Tian, J., Zeng, J., Zhang, Y., Zhang, X., Guo, J., Sun, J., Li, Y., Guo, A. and Li, Y. (2019). Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning. Proc Natl Acad Sci U S A 116(11): 5118-5125. PubMed ID: 30796183
The GABAergic system serves as a vital negative modulator in cognitive functions, such as learning and memory, while the mechanisms governing this inhibitory system remain to be elucidated. In Drosophila, the GABAergic anterior paired lateral (APL) neurons mediate a negative feedback essential for odor discrimination; however, their activity is suppressed by learning via unknown mechanisms. In aversive olfactory learning, a group of dopaminergic (DA) neurons is activated on electric shock (ES) and modulates the Kenyon cells (KCs) in the mushroom body, the center of olfactory learning. This study found that the same group of DA neurons also form functional synaptic connections with the APL neurons, thereby emitting a suppressive signal to the latter through Drosophila dopamine 2-like receptor (DD2R). Knockdown of either DD2R or its downstream molecules in the APL neurons results in impaired olfactory learning at the behavioral level. Results obtained from in vivo functional imaging experiments indicate that this DD2R-dependent DA-to-APL suppression occurs during odor-ES conditioning and discharges the GABAergic inhibition on the KCs specific to the conditioned odor. Moreover, the decrease in odor response of the APL neurons persists to the postconditioning phase, and this change is also absent in DD2R knockdown flies. Taken together, these findings show that DA-to-GABA suppression is essential for restraining the GABAergic inhibition during conditioning, as well as for inducing synaptic modification in this learning circuit. Such circuit mechanisms may play conserved roles in associative learning across species.
Abuin, L., Prieto-Godino, L. L., Pan, H., Gutierrez, C., Huang, L., Jin, R. and Benton, R. (2019). In vivo assembly and trafficking of olfactory ionotropic receptors. BMC Biol 17(1): 34. PubMed ID: 30995910
Ionotropic receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors (iGluRs) that are expressed in diverse peripheral sensory neurons and function in olfaction, taste, hygrosensation and thermosensation. Analogous to the cell biological properties of their synaptic iGluR ancestors, IRs are thought to form heteromeric complexes that localise to the ciliated dendrites of sensory neurons. IR complexes are composed of selectively expressed 'tuning' receptors and one of two broadly expressed co-receptors (IR8a or IR25a). This study identified a sequence in the co-receptor LBD, the 'co-receptor extra loop' (CREL), which is conserved across IR8a and IR25a orthologues but not present in either tuning IRs or iGluRs. The CREL contains a single predicted N-glycosylation site, which bears a sugar modification in recombinantly expressed IR8a. Using the Drosophila olfactory system as an in vivo model, a transgenically encoded IR8a mutant was found in which the CREL that cannot be N-glycosylated is impaired in localisation to cilia in some, though not all, populations of sensory neurons expressing different tuning IRs. This defect can be complemented by the presence of endogenous wild-type IR8a, indicating that IR complexes contain at least two IR8a subunits and that this post-translational modification is dispensable for protein folding or complex assembly. Analysis of the subcellular distribution of the mutant protein suggests that its absence from sensory cilia is due to a failure in exit from the endoplasmic reticulum. CREL N-glycosylation site is likely to be located on the external face of a heterotetrameric IR complex. These data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.
Ando, T., Sekine, S., Inagaki, S., Misaki, K., Badel, L., Moriya, H., Sami, M. M., Itakura, Y., Chihara, T., Kazama, H., Yonemura, S. and Hayashi, S. (2019). Nanopore formation in the cuticle of an insect olfactory sensillum. Curr Biol. PubMed ID: 31006566
Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores, which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. This study shows that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.
Takayanagi-Kiya, S. and Kiya, T. (2019). Activity-dependent visualization and control of neural circuits for courtship behavior in the fly Drosophila melanogaster. Proc Natl Acad Sci U S A 116(12): 5715-5720. PubMed ID: 30837311
Males of Drosophila melanogaster exhibit stereotypic courtship behavior through which they assess potential mates by processing multimodal sensory information. Although previous studies revealed important neural circuits involved in this process, the full picture of circuits that participate in male courtship remains elusive. This study established a genetic tool to visualize or optogenetically reactivate neural circuits activated upon specific behavior, exploiting promoter activity of a neural activity-induced gene Hr38. With this approach, neural circuits activated in the male brain and the ventral nerve cord were visualized when a male interacted with a female. The labeling of neural circuits was additively dependent on inputs from antennae and foreleg tarsi. In addition, neural circuits that express the sex-determining gene fruitless or doublesex were extensively labeled by interaction with a female. Furthermore, optogenetic reactivation of the labeled neural circuits induced courtship posture. With this mapping system, it was found that a fruitless-positive neural cluster aSP2 was labeled when a male interacted with a female, in addition to previously characterized neurons. Silencing of neurons including aSP2 led to frequent interruption of courtship and significant reduction of mating success rate without affecting latency to start courtship, suggesting that these neurons are required for courtship persistency important for successful copulation. Overall, these results demonstrate that activity-dependent labeling can be used as a powerful tool not only in vertebrates, but also in invertebrates, to identify neural circuits regulating innate behavior.
Weiglein, A., Gerstner, F., Mancini, N., Schleyer, M. and Gerber, B. (2019). One-trial learning in larval Drosophila. Learn Mem 26(4): 109-120. PubMed ID: 30898973
Animals of many species are capable of "small data" learning, that is, of learning without repetition. Ghis study introduces larval Drosophila melanogaster as a relatively simple study case for such one-trial learning. Using odor-food associative conditioning, it was first shows that a sugar that is both sweet and nutritious (fructose) and sugars that are only sweet (arabinose) or only nutritious (sorbitol) all support appetitive one-trial learning. The same is the case for the optogenetic activation of a subset of dopaminergic neurons innervating the mushroom body, the memory center of the insects. In contrast, no one-trial learning is observed for an amino acid reward (aspartic acid). As regards the aversive domain, one-trial learning is demonstrated for high-concentration sodium chloride, but is not observed for a bitter tastant (quinine). Second, follow-up, parametric analyses are provided of odor-fructose learning. Specifically,its dependency on the number and duration of training trials was ascertained, as well as the requirements for the behavioral expression of one-trial odor-fructose memory, its temporal stability, and the feasibility of one-trial differential conditioning. The results set the stage for a neurogenetic analysis of one-trial learning and define the requirements for modeling mnemonic processes in the larva.

Yadav, S., Younger, S. H., Zhang, L., Thompson-Peer, K. L., Li, T., Jan, L. Y. and Jan, Y. N. (2019). Glial ensheathment of the somatodendritic compartment regulates sensory neuron structure and activity. Proc Natl Acad Sci U S A 116(11): 5126-5134. PubMed ID: 30804200
Sensory neurons perceive environmental cues and are important of organismal survival. Peripheral sensory neurons interact intimately with glial cells. While the function of axonal ensheathment by glia is well studied, less is known about the functional significance of glial interaction with the somatodendritic compartment of neurons. This study show athat three distinct glia cell types differentially wrap around the axonal and somatodendritic surface of the polymodal dendritic arborization (da) neuron of the Drosophila peripheral nervous system for detection of thermal, mechanical, and light stimuli. Glial cell-specific loss of the chromatin modifier gene dATRX in the subperineurial glial layer was found to lead to selective elimination of somatodendritic glial ensheathment, thus allowing investigation of the function of such ensheathment. Somatodendritic glial ensheathment was found to regulates the morphology of the dendritic arbor, as well as the activity of the sensory neuron, in response to sensory stimuli. Additionally, glial ensheathment of the neuronal soma influences dendritic regeneration after injury.

Friday, April 26th - Gene Regulation

Pherson, M., Misulovin, Z., Gause, M. and Dorsett, D. (2019). Cohesin occupancy and composition at enhancers and promoters are linked to DNA replication origin proximity in Drosophila. Genome Res 29(4):602-612. PubMed ID: 30796039
Cohesin consists of the SMC1-SMC3-Rad21 tripartite ring and the SA protein that interacts with Rad21. The Nipped-B protein loads cohesin topologically around chromosomes to mediate sister chromatid cohesion and facilitate long-range control of gene transcription. It is largely unknown how Nipped-B and cohesin associate specifically with gene promoters and transcriptional enhancers, or how sister chromatid cohesion is established. This study used genome-wide chromatin immunoprecipitation in Drosophila cells to show that Stromalin (SA) and the Fs(1)h (BRD4) BET domain protein help recruit Nipped-B and cohesin to enhancers and DNA replication origins, whereas the MED30 subunit of the Mediator complex directs Nipped-B and Vtd in Drosophila (also known as Rad21) to promoters. All enhancers and their neighboring promoters are close to DNA replication origins and bind SA with proportional levels of cohesin subunits. Most promoters are far from origins and lack SA but bind Nipped-B and Rad21 with subproportional amounts of SMC1, indicating that they bind cohesin rings only part of the time. Genetic data show that Nipped-B and Rad21 function together with Fs(1)h to facilitate Drosophila development. These findings show that Nipped-B and cohesin are differentially targeted to enhancers and promoters, and suggest models for how SA and DNA replication help establish sister chromatid cohesion and facilitate enhancer-promoter communication. They indicate that SA is not an obligatory cohesin subunit but a factor that controls cohesin location on chromosomes.
Shir-Shapira, H., et al. (2019). Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes. PLoS One 14(4): e0215695. PubMed ID: 30998799
The regulation of transcription initiation is critical for developmental and cellular processes. Pol II is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. Previous work has shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. This study examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. This analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. It was further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.
Suzuki, T. K., Koshikawa, S., Kobayashi, I., Uchino, K. and Sezutsu, H. (2019). Modular cis-regulatory logic of yellow gene expression in silkmoth larvae. Insect Mol Biol. PubMed ID: 30737958
Color patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (i.e., yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remains poorly understood. To elucidate this, molecular studies involving dipterans have largely analyzed the cis-regulatory regions of pigmentation genes and have revealed cis-regulatory modularity. This study used well-developed transgenic techniques in Bombyx mori and demonstrated that cis-regulatory modularity controls tissue-specific expression of the yellow gene. First, which body parts are regulated by the yellow gene via black pigmentation were edentified. Then, three discrete regulatory elements driving tissue-specific gene expression in three regions of B. mori larvae were identified. Finally, it was found that there is no apparent sequence conservation of cis-regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans-regulatory landscapes of the yellow gene differ significantly between both taxa. The results of this study confirm that lepidopteran species use cis-regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.
Immarigeon, C., et al. (2019). Drosophila mediator aubunit Med1 is required for GATA-dependent developmental processes: Divergent binding interfaces for conserved coactivator functions. Mol Cell Biol 39(7). PubMed ID: 30670567
DNA-bound transcription factors (TFs) governing developmental gene regulation have been proposed to recruit polymerase II machinery at gene promoters through specific interactions with dedicated subunits of the evolutionarily conserved Mediator (MED) complex. However, whether such MED subunit-specific functions and partnerships have been conserved during evolution has been poorly investigated. To address this issue, this study generated the first Drosophila melanogaster loss-of-function mutants for Med1, known as a specific cofactor for GATA TFs and hormone nuclear receptors in mammals. Med1 was shown to be required for cell proliferation and hematopoietic differentiation depending on the GATA TF Serpent (Srp). Med1 physically binds Srp in cultured cells and in vitro through its conserved GATA zinc finger DNA-binding domain and the divergent Med1 C terminus. Interestingly, GATA-Srp interaction occurs through the longest Med1 isoform, suggesting a functional diversity of MED complex populations. Furthermore, Med1 was shown to act as a coactivator for the GATA factor Pannier during thoracic development. In conclusion, the Med1 requirement for GATA-dependent regulatory processes is a common feature in insects and mammals, although binding interfaces have diverged. Further work in Drosophila should bring valuable insights to fully understand GATA-MED functional partnerships, which probably involve other MED subunits depending on the cellular context.
DeAguero, A. A., Castillo, L., Oas, S. T., Kiani, K., Bryantsev, A. L. and Cripps, R. M. (2019). Regulation of fiber-specific actin expression by the Drosophila SRF ortholog Blistered. Development PubMed ID: 30872277
Serum response factor (SRF) has an established role in controlling actin homeostasis in mammalian cells, yet its role in non-vertebrate muscle development has remained enigmatic. This study demonstrate that the single Drosophila SRF ortholog, termed Blistered (Bs), is expressed in all adult muscles, but Bs is required for muscle organization only in the adult indirect flight muscles. Bs is a direct activator of the flight muscle actin gene, Act88F, via a conserved promoter-proximal binding site. However, Bs only activates Act88F expression in the context of the flight muscle regulatory program provided by the Pbx and Meis orthologs Extradenticle and Homothorax, and appears to function in a similar manner to mammalian SRF in muscle maturation. These studies place Bs in a regulatory framework where it functions to sustain the flight muscle phenotype in Drosophila. These studies uncover an evolutionarily-ancient role for SRF in regulating muscle actin expression, and provide a model for how SRF might function to sustain muscle fate downstream of pioneer factors.
Scholes, C., Biette, K. M., Harden, T. T. and DePace, A. H. (2019). Signal integration by shadow enhancers and enhancer duplications varies across the Drosophila embryo. Cell Rep 26(9): 2407-2418.e2405. PubMed ID: 30811990
Transcription of developmental genes is controlled by multiple enhancers. Frequently, more than one enhancer can activate transcription from the same promoter in the same cells. How is regulatory information from multiple enhancers combined to determine the overall expression output? This study measured nascent transcription driven by a pair of Krüppel shadow enhancers, each enhancer of the pair separately, and each duplicated, using live imaging in Drosophila embryos. This set of constructs allows quantification of the input-output function describing signal integration by two enhancers. Signal integration performed by these shadow enhancers and duplications varies across the expression pattern, implying that how their activities are combined depends on the transcriptional regulators bound to the enhancers in different parts of the embryo. Characterizing signal integration by multiple enhancers is a critical step in developing conceptual and computational models of gene expression at the locus level, where multiple enhancers control transcription together.

Thursday, April 25th - Oogenesis and Spermatogenesis

Hundertmark, T., Kreutz, S., Merle, N., Nist, A., Lamp, B., Stiewe, T., Brehm, A., Renkawitz-Pohl, R. and Rathke, C. (2019). Drosophila melanogaster tPlus3a and tPlus3b ensure full male fertility by regulating transcription of Y-chromosomal, seminal fluid, and heat shock genes. PLoS One 14(3): e0213177. PubMed ID: 30845228
Spermatogenesis in Drosophila is characterized by a specific transcriptional program during the spermatocyte stage. Transcription of thousands of genes is regulated by the interaction of several proteins or complexes, including a tTAF-containing TFIID variant, tMAC, Mediator, and chromatin interactors, e.g., bromodomain proteins. This study addressed how distinct subsets of target genes are selected. The highly similar proteins tPlus3a and tPlus3b (CG31702 and CG31703) that contain a Plus3 domain and are enriched in the testis, mainly in spermatocytes, were. characterized. In tPlus3a and tplus3b deletion mutants generated using the CRISPR/Cas9 system, fertility was severely reduced and sperm showed defects during individualization. tPlus3a and tPlus3b heterodimerized with the bromodomain protein tBRD-1. To elucidate the role of the tPlus3a and tPlus3b proteins in transcriptional regulation, the transcriptomes of tplus3a-tplus3b and tbrd-1 deletion mutants were determined using next-generation sequencing (RNA-seq), and they were compared to that of the wild-type. tPlus3a and tPlus3b positively or negatively regulated the expression of nearly 400 genes; tBRD-1 regulated 1,500 genes. Nearly 200 genes were regulated by both tPlus3a and tPlus3b and tBRD-1. tPlus3a and tPlus3b activated the Y-chromosomal genes kl-3 and kl-5, which indicates that tPlus3a and tPlus3b proteins are required for the function of distinct classes of genes. tPlus3a and tPlus3b and tBRD-1 repress genes relevant for seminal fluid and heat shock. It is hypothesize that tPlus3a and tPlus3b proteins are required to specify the general transcriptional program in spermatocytes.
Barr, J., Charania, S., Gilmutdinov, R., Yakovlev, K., Shidlovskii, Y. and Schedl, P. (2019). The CPEB translational regulator, Orb, functions together with Par proteins to polarize the Drosophila oocyte. PLoS Genet 15(3): e1008012. PubMed ID: 30865627
orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. This study reports that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. Repolarization of the oocyte also requires orb and orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.
Li, Z., Yang, F., Xuan, Y., Xi, R. and Zhao, R. (2019). Pelota-interacting G protein Hbs1 is required for spermatogenesis in Drosophila. Sci Rep 9(1): 3226. PubMed ID: 30824860
Hbs1, which is homologous to the GTPase eRF3, is a small G protein implicated in mRNA quality control. It interacts with a translation-release factor 1-like protein Dom34/Pelota to direct decay of mRNAs with ribosomal stalls. Although both proteins are evolutionarily conserved in eukaryotes, the biological function of Hbs1 in multicellular organisms is yet to be characterized. In Drosophila, pelota is essential for the progression through meiosis during spermatogenesis and germline stem cell maintenance. Homozygous Hbs1 mutant flies are viable, female-fertile, but male-sterile, which is due to defects in meiosis and spermatid individualization, phenotypes that are also observed in pelota hypomorphic mutants. In contrast, Hbs1 mutants have no obvious defects in germline stem cell maintenance. Hbs1 genetically interacts with pelota during spermatid individualization. Furthermore, Pelota with a point mutation on the putative Hbs1-binding site cannot substitute the wild type protein for normal spermatogenesis. These data suggest that Pelota forms a complex with Hbs1 to regulate multiple processes during spermatogenesis. The results reveal a specific requirement of Hbs1 in male gametogenesis in Drosophila and indicate an essential role for the RNA surveillance complex Pelota-Hbs1 in spermatogenesis, a function that could be conserved in mammals.
Ben-Zvi, D. S. and Volk, T. (2019). Escort cell encapsulation of Drosophila germline cells is maintained by Irre cell recognition module proteins. Biol Open 8(3). PubMed ID: 30837217
Differentiation of germline stem cells (GSCs) in the Drosophila ovary is induced by somatic escort cells (ECs), which extend membrane protrusions encapsulating the germline cells (GCs). Germline encapsulation requires activated epidermal growth factor receptor (Egfr) signaling within the ECs, following secretion of its ligands from the GCs. This study shows that the conserved family of irre cell recognition module (IRM) proteins is essential for GC encapsulation by ECs, with a requirement for roughest (rst) and kin of irre (kirre) in the germline and for sticks and stones (sns) and hibris (hbs) in ECs. In the absence of IRM components in their respective cell types, EC extensions are reduced concomitantly with a decrease in Egfr signaling in these cells. Reintroducing either activated Egfr in the ECs, or overexpressing its ligand Spitz (Spi) from the germline, rescued the requirement for IRM proteins in both cell types. These experiments introduce novel essential components, the IRM proteins, into the process of inductive interactions between GCs and ECs, and imply that IRM-mediated activity is required upstream of the Egfr signaling.
Yu, J., Chen, B., Zheng, B., Qiao, C., Chen, X., Yan, Y., Luan, X., Xie, B., Liu, J., Shen, C., He, Z., Hu, X., Liu, M., Li, H., Shao, Q. and Fang, J. (2019). ATP synthase is required for male fertility and germ cell maturation in Drosophila testes. Mol Med Rep 19(3): 1561-1570. PubMed ID: 30628672
Germ cell maturation is essential for spermatogenesis and testis homeostasis. ATP synthase serves significant roles in energy storage in germ cell survival and is catalyzed by alterations in the mitochondrial membrane proton concentration. The intrinsic cellular mechanisms governing stem cell maturation remain largely unknown. In the present study, in vivo RNA interference (RNAi) screening of major ATP synthase subunits was performed, and the function of ATP synthase for male fertility and spermatogenesis in Drosophila was explored. A Upstream Activation Sequence/Gal4 transcription factor system was used to knock down gene expression in specific cell types, and immunofluorescence staining was conducted to assess the roles of ATP synthase subunits in Drosophila testes. It was identified that knockdown of ATP synthase resulted in male infertility and abnormal spermatogenesis in Drosophila testes. In addition, knockdown of the ATP synthase beta subunit in germ cells resulted in defects in male infertility and germ cell maturation, while the hub and cyst cell populations were maintained. Other major ATP synthase subunits were also examined and similar phenotypes in Drosophila testes were identified. Taken together, the data from the present study revealed that ATP synthase serves important roles for male fertility during spermatogenesis by regulating germ cell maturation in Drosophila testes.
Jeong, E. B., Jeong, S. S., Cho, E. and Kim, E. Y. (2019). Makorin 1 is required for Drosophila oogenesis by regulating insulin/Tor signaling. PLoS One 14(4): e0215688. PubMed ID: 31009498
Reproduction is a process that is extremely sensitive to changes in nutritional status. The nutritional control of oogenesis via insulin signaling has been reported; however, the mechanism underlying its sensitivity and tissue specificity has not been elucidated. This study determined that Drosophila Makorin RING finger protein 1 gene (Mkrn1) functions in the metabolic regulation of oogenesis. Mkrn1 was endogenously expressed at high levels in ovaries and Mkrn1 knockout resulted in female sterility. Mkrn1-null egg chambers were previtellogenic without egg production. FLP-FRT mosaic analysis revealed that Mkrn1 is essential in germline cells, but not follicle cells, for ovarian function. As well, AKT phosphorylation via insulin signaling was greatly reduced in the germline cells, but not the follicle cells, of the mutant clones in the ovaries. Furthermore, protein-rich diet elevated Mkrn1 protein levels, without increased mRNA levels. The p-AKT and p-S6K levels, downstream targets of insulin/Tor signaling, were significantly increased by a nutrient-rich diet in wild-type ovaries whereas those were low in Mkrn1exS compared to wild-type ovaries. Taken together, these results suggest that nutrient availability upregulates the Mkrn1 protein, which acts as a positive regulator of insulin signaling to confer sensitivity and tissue specificity in the ovaries for proper oogenesis based on nutritional status.

Wednesday, April 24th - Cell Cycle

Moreno-Moreno, O., Torras-Llort, M. and Azorin, F. (2019). The E3-ligases SCFPpa and APC/CCdh1 co-operate to regulate CENP-ACID expression across the cell cycle. Nucleic Acids Res. PubMed ID: 30753559
Centromere identity is determined by the specific deposition of CENP-A, a histone H3 variant localizing exclusively at centromeres. Increased CENP-A expression, which is a frequent event in cancer, causes mislocalization, ectopic kinetochore assembly and genomic instability. Proteolysis regulates CENP-A expression and prevents its misincorporation across chromatin. How proteolysis restricts CENP-A localization to centromeres is not well understood. ^hie e6ury reports that, in Drosophila, CENP-ACID expression levels are regulated throughout the cell cycle by the combined action of SCFPpa and APC/CCdh1. SCFPpa regulates CENP-ACID expression in G1 and, importantly, in S-phase preventing its promiscuous incorporation across chromatin during replication. In G1, CENP-ACID expression is also regulated by APC/CCdh1. This study also shows that Cal1, the specific chaperone that deposits CENP-ACID at centromeres, protects CENP-ACID from SCFPpa-mediated degradation but not from APC/CCdh1-mediated degradation. These results suggest that, whereas SCFPpa targets the fraction of CENP-ACID that is not in complex with Cal1, APC/CCdh1 mediates also degradation of the Cal1-CENP-ACID complex and, thus, likely contributes to the regulation of centromeric CENP-Asup>CID deposition.
Liu, B., Winkler, F., Herde, M., Witte, C. P. and Grosshans, J. (2019). A link between deoxyribonucleotide metabolites and embryonic cell-cycle control. Curr Biol. PubMed ID: 30880011
The egg contains maternal RNAs and proteins, which have instrumental functions in patterning and morphogenesis. Besides these, the egg also contains metabolites, whose developmental functions have been little investigated. For example, the rapid increase of DNA content during the fast embryonic cell cycles poses high demands on the supply of deoxyribonucleotides (dNTPs), which may be synthesized in the embryo or maternally provided. This study analyzed the role of dNTP in early Drosophila embryos. dNTP levels initially decreased about 2-fold before reaching stable levels at the transition from syncytial to cellular blastoderm. Employing a mutant of the metabolic enzyme serine hydroxymethyl transferase (SHMT), which is impaired in the embryonic synthesis of deoxythymidine triphosphate (dTTP), it was found that the maternal supply of dTTP was specifically depleted by interphase 13. SHMT mutants showed persistent S phase, replication stress, and a checkpoint-dependent cell-cycle arrest in NC13, depending on the loss of dTTP. The cell-cycle arrest in SHMT mutants was suppressed by reduced zygotic transcription. Consistent with the requirement of dTTP for cell-cycle progression, increased dNTP levels accelerated the cell cycle in embryos lacking zygotic transcription. A model is proposed that both a limiting dNTP supply and interference of zygotic transcription with DNA replication elicit DNA replication stress and checkpoint activation. This study reveals a specific mechanism of how dNTP metabolites contribute to the embryonic cell-cycle control.
Betsch, L., Boltz, V., Brioudes, F., Pontier, G., Girard, V., Savarin, J., Wipperman, B., Chambrier, P., Tissot, N., Benhamed, M., Mollereau, B., Raynaud, C., Bendahmane, M. and Szecsi, J. (2019). TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals. PLoS Genet 15(1): e1007899. PubMed ID: 30695029
Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, these data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP's role in organ development and disorders associated with TCTP miss-expression.
Carim, S. C., Ben El Kadhi, K., Yan, G., Sweeney, S. T., Hickson, G. R., Carreno, S. and Lowe, M. (2019). IPIP27 coordinates PtdIns(4,5)P2 homeostasis for successful cytokinesis. Curr Biol 29(5): 775-789. PubMed ID: 30799246
During cytokinesis, an actomyosin contractile ring drives the separation of the two daughter cells. A key molecule in this process is the inositol lipid PtdIns(4,5)P2, which recruits numerous factors to the equatorial region for contractile ring assembly. Despite the importance of PtdIns(4,5)P2 in cytokinesis, the regulation of this lipid in cell division remains poorly understood. This study identified a role for IPIP27 in mediating cellular PtdIns(4,5)P2 homeostasis. IPIP27 scaffolds the inositol phosphatase oculocerebrorenal syndrome of Lowe (OCRL) by coupling it to endocytic BAR domain proteins. Loss of IPIP27 causes accumulation of PtdIns(4,5)P2 on aberrant endomembrane vacuoles, mislocalization of the cytokinetic machinery, and extensive cortical membrane blebbing. This phenotype is observed in Drosophila and human cells and can result in cytokinesis failure. This study has therefore identified IPIP27 as a key modulator of cellular PtdIns(4,5)P2 homeostasis required for normal cytokinesis. The results indicate that scaffolding of inositol phosphatase activity is critical for maintaining PtdIns(4,5)P2 homeostasis and highlight a critical role for this process in cell division.

Tuesday, April 23rd - Chromatin

Parey, E. and Crombach, A. (2019). Evolution of the Drosophila melanogaster chromatin landscape and its associated proteins. Genome Biol Evol 11(3): 660-677. PubMed ID: 30689829
In the nucleus of eukaryotic cells, genomic DNA associates with numerous protein complexes and RNAs, forming the chromatin landscape. Through a genome-wide study of chromatin-associated proteins in Drosophila cells, five major chromatin types were identified as a refinement of the traditional binary division into hetero- and euchromatin. These five types were given color names in reference to the Greek word chroma. They are defined by distinct but overlapping combinations of proteins and differ in biological and biochemical properties, including transcriptional activity, replication timing, and histone modifications. This work assesses the evolutionary relationships of chromatin-associated proteins and present an integrated view of the evolution and conservation of the fruit fly Drosophila melanogaster chromatin landscape. Homology prediction across a wide range of species was combined with gene age inference methods to determine the origin of each chromatin-associated protein. This provides insight into the evolution of the different chromatin types. The results indicate that for the euchromatic types, YELLOW and RED, young associated proteins are more specialized than old ones; and for genes found in either chromatin type, intron/exon structure is lineage-specific. Next, evidence is provided that a subset of GREEN-associated proteins is involved in a centromere drive in D. melanogaster. The results on BLUE chromatin support the hypothesis that the emergence of Polycomb Group proteins is linked to eukaryotic multicellularity. In light of these results, how the regulatory complexification of chromatin links to the origins of eukaryotic multicellularity is discussed.
Riahi, H., Brekelmans, C., Foriel, S., Merkling, S. H., Lyons, T. A., Itskov, P. M., Kleefstra, T., Ribeiro, C., van Rij, R. P., Kramer, J. M. and Schenck, A. (2019). The histone methyltransferase G9a regulates tolerance to oxidative stress-induced energy consumption.. PLoS Biol 17(3): e2006146. PubMed ID: 30860988
The mechanisms coordinating energy consumption during stress response in multicellular organisms are not well understood. This study shows that loss of the epigenetic regulator G9a in Drosophila causes a shift in the transcriptional and metabolic responses to oxidative stress (OS) that leads to decreased survival time upon feeding the xenobiotic paraquat. During OS exposure, G9a mutants show overactivation of stress response genes, rapid depletion of glycogen, and inability to access lipid energy stores. The OS survival deficiency of G9a mutants can be rescued by a high-sugar diet. Control flies also show improved OS survival when fed a high-sugar diet, suggesting that energy availability is generally a limiting factor for OS tolerance. Directly limiting access to glycogen stores by knocking down glycogen phosphorylase recapitulates the OS-induced survival defects of G9a mutants. It is proposed that G9a mutants are sensitive to stress because they experience a net reduction in available energy due to (1) rapid glycogen use, (2) an inability to access lipid energy stores, and (3) an overinduced transcriptional response to stress that further exacerbates energy demands. This suggests that G9a acts as a critical regulatory hub between the transcriptional and metabolic responses to OS. These findings, together with recent studies that established a role for G9a in hypoxia resistance in cancer cell lines, suggest that G9a is of wide importance in controlling the cellular and organismal response to multiple types of stress.
Saha, P., Sowpati, D. T. and Mishra, R. K. (2019). Epigenomic and genomic landscape of Drosophila melanogaster heterochromatic genes. Genomics 111(2): 177-185. PubMed ID: 29432976
Heterochromatin is associated with transcriptional repression. In contrast, several genes in the pericentromeric regions of Drosophila melanogaster are dependent on this heterochromatic environment for their expression. This study presents a comprehensive analysis of the epigenetic landscape of heterochromatic genes across all the developmental stages of Drosophila using the available histone modification and expression data from modENCODE. Heterochromatic genes were found to exhibit combinations of active and inactive histone marks that correspond to their level of expression during development. Nuclear Matrix Associated Regions (MARs) are prominently present in the intergenic regions of heterochromatic genes during embryonic stages suggesting their plausible role in pericentromeric genome organization. Taken together, this meta-analysis of the various genomic datasets suggest that the epigenomic and genomic landscape of the heterochromatic genes are distinct which could be contributing to their unusual regulatory features as opposed to the surrounding heterochromatin, which is repressive in nature.
Seller, C. A., Cho, C. Y. and O'Farrell, P. H. (2019). Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila. Genes Dev. PubMed ID: 30808658
Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naive genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/trimethylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila embryo, the heterochromatin lacks these modifications, which appear only later, when rapid embryonic cell cycles slow down at the midblastula transition (MBT). This study focused on the initial steps restoring heterochromatic modifications in the embryo. The JabbaTrap, a technique for inactivating maternally provided proteins in embryos, is described. Using the JabbaTrap, a major requirement was revealed for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. Live imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase but then dissociates in mitosis, and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. It is proposed that developmental slowing of the cell cycle times embryonic heterochromatin formation.
Rowley, M. J., Lyu, X., Rana, V., Ando-Kuri, M., Karns, R., Bosco, G. and Corces, V. G. (2019). Condensin II counteracts Cohesin and RNA Polymerase II in the establishment of 3D chromatin organization. Cell Rep 26(11): 2890-2903.e2893. PubMed ID: 30865881
Interaction domains in Drosophila chromosomes form by segregation of active and inactive chromatin in the absence of CTCF loops, but the role of transcription versus other architectural proteins in chromatin organization is unclear. This study finds that positioning of RNAPII via transcription elongation is essential in the formation of gene loops, which in turn interact to form compartmental domains. Inhibition of transcription elongation or depletion of cohesin decreases gene looping and formation of active compartmental domains. In contrast, depletion of condensin II, which also localizes to active chromatin, causes increased gene looping, formation of compartmental domains, and stronger intra-chromosomal compartmental interactions. Condensin II has a similar role in maintaining inter-chromosomal interactions responsible for pairing between homologous chromosomes, whereas inhibition of transcription elongation or cohesin depletion has little effect on homolog pairing. The results suggest distinct roles for cohesin and condensin II in the establishment of 3D nuclear organization in Drosophila.
Ulianov, S. V., Doronin, S. A., Khrameeva, E. E., Kos, P. I., Luzhin, A. V., Starikov, S. S., Galitsyna, A. A., Nenasheva, V. V., Ilyin, A. A., Flyamer, I. M., Mikhaleva, E. A., Logacheva, M. D., Gelfand, M. S., Chertovich, A. V., Gavrilov, A. A., Razin, S. V. and Shevelyov, Y. Y. (2019). Nuclear lamina integrity is required for proper spatial organization of chromatin in Drosophila. Nat Commun 10(1): 1176. PubMed ID: 30862957
How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. This study shows that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, these findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes.

Monday, April 22nd - Autophagy and Apoptosis

Vincow, E. S., Thomas, R. E., Merrihew, G. E., Shulman, N. J., Bammler, T. K., MacDonald, J. W., MacCoss, M. J. and Pallanck, L. J. (2019). Autophagy accounts for approximately one-third of mitochondrial protein turnover and is protein selective. Autophagy: 1-14. PubMed ID: 30865561
The destruction of mitochondria through macroautophagy (autophagy) has been recognised as a major route of mitochondrial protein degradation. Autophagy was originally thought to degrade all mitochondrial proteins at the same rate, but recent work suggests that mitochondrial autophagy may be protein selective. To investigate these questions, a proteomics-based approach was used in the fruit fly Drosophila melanogaster, comparing mitochondrial protein turnover rates in autophagy-deficient Atg7 mutants and controls. ~35% of mitochondrial protein turnover occurred via autophagy. Similar analyses using parkin mutants revealed that parkin-dependent mitophagy accounted for ~25% of mitochondrial protein turnover, suggesting that most mitochondrial autophagy specifically eliminates dysfunctional mitochondria. The results were incompatible with uniform autophagic turnover of mitochondrial proteins and consistent with protein-selective autophagy. In particular, the autophagic turnover rates of individual mitochondrial proteins varied widely, and only a small amount of the variation could be attributed to tissue differences in mitochondrial composition and autophagy rate. Furthermore, analyses comparing autophagy-deficient and control human fibroblasts revealed diverse autophagy-dependent turnover rates even in homogeneous cells. In summary, this work indicates that autophagy acts selectively on mitochondrial proteins, and that most mitochondrial protein turnover occurs through non-autophagic processes.
Sun, Y., Zhang, D., Li, C., Huang, J., Li, W., Qiu, Y., Mao, A., Zhou, M. and Xue, L. (2019). Lic regulates JNK-mediated cell death in Drosophila. Cell Prolif: e12593. PubMed ID: 30847993
The evolutionary conserved JNK pathway plays crucial role in cell death, yet factors that modulate this signalling have not been fully disclosed. This study aimed to identify additional factors that regulate JNK signalling in cell death, and characterize the underlying mechanisms. Drosophila were raised on standard media, and cross was carried out at 25 degrees C. The Gal4/UAS system was used to express proteins or RNAi in a specific temporal and spatial pattern. Gene expression was revealed by GFP fluorescence, X-gal staining or immunostaining of 3rd instar larval eye and wing discs. Cell death was visualized by acridine orange (AO) staining. Images of fly eyes and wings were taken by OLYMPUS microscopes. licorne (lic) encoding the Drosophila MKK3 was shown to be an essential regulator of JNK-mediated cell death. Firstly, loss of lic suppressed ectopic Egr-triggered JNK activation and cell death in eye and wing development. Secondary, lic is necessary for loss-of-cell polarity-induced, physiological JNK-dependent cell death in wing development. Thirdly, Lic overexpression is sufficient to initiate JNK-mediated cell death in developing eyes and wings. Furthermore, ectopic Lic activates JNK signalling by promoting JNK phosphorylation. Finally, genetic epistatic analysis confirmed that Lic acts in parallel with Hep in the Egr-JNK pathway. This study not only identified Lic as a novel component of the JNK signalling, but also disclosed the crucial roles and mechanism of Lic in cell death.
Texada, M. J., Malita, A., Christensen, C. F., Dall, K. B., Faergeman, N. J., Nagy, S., Halberg, K. A. and Rewitz, K. (2019). Autophagy-mediated cholesterol trafficking controls steroid production. Dev Cell 48(5): 659-671.e654. PubMed ID: 30799225
Steroid hormones are important signaling molecules that regulate growth and drive the development of many cancers. These factors act as long-range signals that systemically regulate the growth of the entire organism, whereas the Hippo/Warts tumor-suppressor pathway acts locally to limit organ growth. This study shows that autophagy, a pathway that mediates the degradation of cellular components, also controls steroid production. This process is regulated by Warts (in mammals, LATS1/2) signaling, via its effector microRNA bantam, in response to nutrients. Specifically, autophagy-mediated mobilization and trafficking of the steroid precursor cholesterol from intracellular stores controls the production of the Drosophila steroid ecdysone. Furthermore, it was also shown that bantam regulates this process via the ecdysone receptor and Tor signaling, identifying pathways through which bantam regulates autophagy and growth. The Warts pathway thus promotes nutrient-dependent systemic growth during development by autophagy-dependent steroid hormone regulation (ASHR). These findings uncover an autophagic trafficking mechanism that regulates steroid production.
Wang, X. C., Liu, Z. and Jin, L. H. (2019). Drosophila jumu modulates apoptosis via a JNK-dependent pathway and is required for other processes in wing development. Apoptosis. PubMed ID: 30796611
Previous studies in several model organisms have revealed that members of the Forkhead (Fkh) transcription factor family have multiple functions. Drosophila Jumeau (Jumu), a member of this family, participates in cardiogenesis, hematopoiesis and immune system homeostasis. This study shows that loss of jumu function positively regulates or triggers apoptosis via a JNK-dependent pathway in wing development. jumu mutants showed reduced wing size and increased apoptosis. Moreover, a loss was observed of the anterior cross vein (ACV) phenotype that was similar to that observed in wings in which JNK signaling has been ectopically activated. The JNK signaling markers puckered (puc) and p-JNK were also significantly increased in the wing discs of jumu mutants. In addition, apoptosis induced by the loss of jumu was rescued by knocking down JNK, indicating a role for JNK in reducing jumu-induced apoptosis. Jumu could also control wing margin development via the positive regulation of cut expression, and the observed wing margin defect did not result from a loss of jumu-induced apoptosis. Further, jumu deficiency in the pupal wing could induce multiple wing hairs via a Rho1-mediated planar cell polarity pathway, but abnormal Rho1 expression was not why jumu loss induced apoptosis via a JNK-dependent pathway in wing discs.
Robin, M., Issa, A. R., Santos, C. C., Napoletano, F., Petitgas, C., Chatelain, G., Ruby, M., Walter, L., Birman, S., Domingos, P. M., Calvi, B. R. and Mollereau, B. (2019). Drosophila p53 integrates the antagonism between autophagy and apoptosis in response to stress. Autophagy 15(5): 771-784. PubMed ID: 30563404
The tumor suppressor TP53/p53 is a known regulator of apoptosis and macroautophagy/autophagy. However, the molecular mechanism by which TP53 regulates two apparently incompatible processes remains unknown. This study found that Drosophila lacking p53 displayed impaired autophagic flux, higher caspase activation and mortality in response to oxidative stress compared with wild-type flies. Moreover, autophagy and apoptosis were differentially regulated by the p53 (p53B) and DeltaNp53 (p53A) isoforms: while the former induced autophagy in differentiated neurons, which protected against cell death, the latter inhibited autophagy by activating the caspases Dronc, Drice, and Dcp-1. These results demonstrate that the differential use of p53 isoforms combined with the antagonism between apoptosis and autophagy ensures the generation of an appropriate p53 biological response to stress.
Xu, T., Denton, D. and Kumar, S. (2019). Hedgehog and Wingless signaling are not essential for autophagy-dependent cell death. Biochem Pharmacol 162: 3-13. PubMed ID: 30879494
Autophagy-dependent cell death is a distinct mode of regulated cell death required in a context specific manner. One of the best validated genetic models of autophagy-dependent cell death is the removal of the Drosophila larval midgut during larval-pupal transition. Previous work has shown that down-regulation of growth signaling is essential for autophagy induction and larval midgut degradation. Sustained growth signaling through Ras and PI3K blocks autophagy and consequently inhibits midgut degradation. In addition, the morphogen Dpp plays an important role in regulating the correct timing of midgut degradation. This study explored the potential roles of Hh and Wg signaling in autophagy-dependent midgut cell death. Hh and Wg signaling are not involved in the regulation of autophagy-dependent cell death. However, surprisingly one key component of these pathways, the Drosophila Glycogen Synthase Kinase 3, Shaggy (Sgg), may regulate midgut cell size independent of Hh and Wg signaling.

Friday, April 19th - Adult Neural Development and Function

Kasture, A. S., Bartel, D., Steinkellner, T., Sucic, S., Hummel, T. and Freissmuth, M. (2019). Distinct contribution of axonal and somatodendritic serotonin transporters in Drosophila olfaction. Neuropharmacology. PubMed ID: 30851308
The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. This study introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting - otherwise fully functional - hSERT-R(607)A on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-R(599)A led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. The possible physiological role of somatodendritic SERT was explored by comparing flies harboring wild type SERT and dSERT-R(599)A in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R599A restored ethanol preference. In contrast, dSERT-R599A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. It is concluded that, in CSD neurons, (i) somatodendritic SERT supports ethanol attraction, (ii) axonal SERT specifies ethanol aversion, (iii) the effect of axonal SERT can override that of somatodendritic SERT. These observations demonstrate a distinct biological role of somatodendritic and axonal serotonin transport.
Schutzler, N., Girwert, C., Hugli, I., Mohana, G., Roignant, J. Y., Ryglewski, S. and Duch, C. (2019). Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state. Proc Natl Acad Sci U S A 116(9): 3805-3810. PubMed ID: 30808766
Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. This study shows that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyramine(honoka) receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. It was further shown that the level of tyramine-beta-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH.
Mohamed, A. A. M., Retzke, T., Das Chakraborty, S., Fabian, B., Hansson, B. S., Knaden, M. and Sachse, S. (2019). Odor mixtures of opposing valence unveil inter-glomerular crosstalk in the Drosophila antennal lobe. Nat Commun 10(1): 1201. PubMed ID: 30867415
Evaluating odor blends in sensory processing is a crucial step for signal recognition and execution of behavioral decisions. Using behavioral assays and 2-photon imaging, this study has characterized the neural and behavioral correlates of mixture perception in the olfactory system of Drosophila. Mixtures of odors with opposing valences elicit strong inhibition in certain attractant-responsive input channels. This inhibition correlates with reduced behavioral attraction. Defined subsets of GABAergic interneurons provide the neuronal substrate of this computation at pre- and postsynaptic loci via GABAB- and GABAA receptors, respectively. Intriguingly, manipulation of single input channels by silencing and optogenetic activation unveils a glomerulus-specific crosstalk between the attractant- and repellent-responsive circuits. This inhibitory interaction biases the behavioral output. Such a form of selective lateral inhibition represents a crucial neuronal mechanism in the processing of conflicting sensory information.
Pahl, M. C., Doyle, S. E. and Siegrist, S. E. (2019). E93 integrates neuroblast intrinsic state with developmental time to terminate MB neurogenesis via autophagy. Curr Biol 29(5): 750-762. PubMed ID: 30773368
Most neurogenesis occurs during development, driven by the cell divisions of neural stem cells (NSCs). This study used Drosophila to understand how neurogenesis terminates once development is complete, a process critical for neural circuit formation. E93 was identified as a steroid-hormone-induced transcription factor that downregulates phosphatidylinositol 3-kinase (PI3K) levels to activate autophagy for elimination of mushroom body (MB) neuroblasts. MB neuroblasts are a subset of Drosophila NSCs that generate neurons important for memory and learning. MB neurogenesis extends into adulthood when E93 is reduced and terminates prematurely when E93 is overexpressed. E93 is expressed in MB neuroblasts during later stages of pupal development only, which includes the time when MB neuroblasts normally terminate their divisions. Cell intrinsic Imp and Syp temporal factors regulate timing of E93 expression in MB neuroblasts, and extrinsic steroid hormone receptor (EcR) activation boosts E93 levels high for termination. Imp inhibits premature expression of E93 in a Syp-dependent manner, and Syp positively regulates E93 to promote neurogenesis termination. Imp and Syp together with E93 form a temporal cassette, which consequently links early developmental neurogenesis with termination. Altogether, E93 functions as a late-acting temporal factor integrating extrinsic hormonal cues linked to developmental timing with neuroblast intrinsic temporal cues to precisely time neurogenesis ending during development.
Jorg, D. J., Caygill, E. E., Hakes, A. E., Contreras, E. G., Brand, A. H. and Simons, B. D. (2019). The proneural wave in the Drosophila optic lobe is driven by an excitable reaction-diffusion mechanism. Elife 8. PubMed ID: 30794154
In living organisms, self-organised waves of signalling activity propagate spatiotemporal information within tissues. During the development of the largest component of the visual processing centre of the Drosophila brain, a travelling wave of proneural gene expression initiates neurogenesis in the larval optic lobe primordium and drives the sequential transition of neuroepithelial cells into neuroblasts. This study proposes that this 'proneural wave' is driven by an excitable reaction-diffusion system involving epidermal growth factor receptor (EGFR) signalling interacting with the proneural gene l'sc. Within this framework, a propagating transition zone emerges from molecular feedback and diffusion. Ectopic activation of EGFR signalling in clones within the neuroepithelium demonstrates that a transition wave can be excited anywhere in the tissue by inducing signalling activity, consistent with a key prediction of the model. This model illuminates the physical and molecular underpinnings of proneural wave progression and suggests a generic mechanism for regulating the sequential differentiation of tissues.
Stratmann, J., Ekman, H. and Thor, S. (2019). A branching gene regulatory network dictating different aspects of a neuronal cell identity. Development 146(6). PubMed ID: 30837222
The nervous system displays a daunting cellular diversity. Neuronal subtypes differ from each other in several aspects, including their neurotransmitter expression and axon projection. These aspects can converge, but can also diverge, such that neurons expressing the same neurotransmitter may project axons to different targets. It is not well understood how regulatory programs converge/diverge to associate/dissociate different cell fate features. Studies of the Drosophila Tv1 neurons have identified a regulatory cascade, ladybird early-->collier-->apterous/eyes absent-->dimmed, that specifies Tv1 neurotransmitter expression. Genetic and transcriptome analysis was conducted to address how other aspects of Tv1 cell fate are governed. An initiator terminal selector gene triggers a feedforward loop that branches into different subroutines, each of which establishes different features of this one unique neuronal cell fate.

Thursday, April 18th

Ong, K., Collier, C. and DiNardo, S. (2019). Multiple feedback mechanisms fine-tune Rho signaling to regulate morphogenetic outcomes. J Cell Sci. PubMed ID: 30872456
Rho signaling is a conserved mechanism for generating forces through activation of contractile actomyosin. How this pathway can produce different cell morphologies is poorly understood. In the Drosophila embryonic epithelium, this study investigated how Rho signaling controls force asymmetry to drive morphogenesis. A distinct morphogenetic process termed "alignment" was studied. This process results in striking columns of rectilinear cells connected by aligned cell-cell contacts. This was found to be driven by contractile actomyosin cables that elevate tension along aligning interfaces. The data show that polarization of Rho effectors, ROK and Dia, direct formation of these cables. Constitutive activation of these effectors causes aligning cells to instead invaginate. This suggests that moderating Rho signaling is essential to producing the aligned geometry. Therefore, tests were performed for feedback that could fine-tune Rho signaling. It was discovered that F-actin exerts negative feedback on multiple nodes in the pathway. Further, evidence is presented that suggests that ROK in part mediates feedback from F-actin to Rho in a Myo-II-independent manner. Collectively, this work suggests that multiple feedback mechanisms regulate Rho signaling, which may account for diverse morphological outcomes.
Ri, H., Lee, J., Sonn, J. Y., Yoo, E., Lim, C. and Choe, J. (2019). Drosophila CrebB is a substrate of the nonsense-mediated mRNA decay pathway that sustains circadian behaviors. Mol Cells. PubMed ID: 30841026
This study reports that Drosophila nonsense-mediated mRNA decay (NMD) pathway acts in a subset of circadian pacemaker neurons to maintain robust 24h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide Pigment-Dispersing Factor (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. It was further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.
Sohr, A., Du, L., Wang, R., Lin, L. and Roy, S. (2019). Drosophila FGF cleavage is required for efficient intracellular sorting and intercellular dispersal. J Cell Biol. PubMed ID: 30808704
How morphogenetic signals are prepared for intercellular dispersal and signaling is fundamental to the understanding of tissue morphogenesis. This study discovered an intracellular mechanism that prepares Drosophila melanogaster FGF Branchless (Bnl) for cytoneme-mediated intercellular dispersal during the development of the larval Air-Sac-Primordium (ASP). Wing-disc cells express Bnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface, where it is received by cytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is mistargeted to the apical side, reducing its bioavailability. Since Bnl signaling levels feedback control cytoneme production in the ASP, the reduced availability of mutant Bnl on the source basal surface decreases ASP cytoneme numbers, leading to a reduced range of signal/signaling gradient and impaired ASP growth. Thus, enzymatic cleavage ensures polarized intracellular sorting and availability of Bnl to its signaling site, thereby determining its tissue-specific intercellular dispersal and signaling range.
Ogura, Y., Sami, M. M., Wada, H. and Hayashi, S. (2019). Automated FRET quantification reveals distinct subcellular ERK activation kinetics in response to graded EGFR signaling in Drosophila. Genes Cells. PubMed ID: 30851218
Threshold responses to an activity gradient allow a single signaling pathway to yield multiple outcomes. Extracellular signal-regulated kinase (ERK) is one such signal, which couples receptor tyrosine kinase signaling with multiple cellular responses in various developmental processes. Recent advances in the development of fluorescent biosensors for live imaging have enabled the signaling activities accompanying embryonic development to be monitored in real time. This study used an automated computational program to quantify the signals of a fluorescence resonance energy transfer (FRET) reporter for activated ERK, and this system was used to monitor the spatio-temporal dynamics of ERK during neuroectoderm patterning in Drosophila embryos. The cytoplasmic and nuclear ERK activity gradients were found to exhibit distinct kinetics in response to epidermal growth factor (EGF) receptor activation. The ERK activation patterns implied that the cytoplasmic ERK activity is modulated into a threshold response in the nucleus.
Lu, Y., Yao, Y. and Li, Z. (2019). Ectopic Dpp signaling promotes stem cell competition through EGFR signaling in the Drosophila testis. Sci Rep 9(1): 6118. PubMed ID: 30992503
Stem cell competition could select the fittest stem cells and potentially control tumorigenesis. However, little is known about the underlying molecular mechanisms. This study finds that that ectopic Decapentaplegic (Dpp) signal activation by expressing a constitutively active form of Thickveins (Tkv(CA)) in cyst stem cells (CySCs) leads to competition between CySCs and germline stem cells (GSCs) for niche occupancy and GSC loss. GSCs are displaced from the niche and undergo differentiation. Interestingly, it was found that induction of Tkv(CA) results in elevated expression of vein, which further activates Epidermal Growth Factor Receptor (EGFR) signaling in CySCs to promote their proliferation and compete GSCs out of the niche. These findings elucidate the important role of Dpp signaling in regulating stem cell competition and tumorigenesis, which could be shed light on tumorigenesis and cancer treatment in mammals.
Powers, N. and Srivastava, A. (2019). JAK/STAT signaling is involved in air sac primordium development of Drosophila melanogaster. FEBS Lett. PubMed ID: 30854626
The dorsal thoracic air sacs in fruit flies (Drosophila melanogaster) are functionally and developmentally comparable to human lungs. The progenitors of these structures, air sac primordia (ASPs), invasively propagate into wing imaginal disks, employing mechanisms similar to those that promote metastasis in malignant tumors. This study investigated whether Janus kinase/signal transducer and activator of transcription JAK/STAT signaling plays a role in the directed morphogenesis of ASPs. JAK/STAT signaling was found to occur in ASP tip cells and misexpression of core components in the JAK/STAT signaling cascade significantly impedes ASP development. Upd2 was identified as an activating ligand for JAK/STAT activity in the ASP. Together, these data constitute a considerable step forward in understanding the role of JAK/STAT signaling in ASPs and similar structures in mammalian models.

Wednesday, April 17th - Apoptosis and Autophagy

Poulton, J. S., McKay, D. J. and Peifer, M. (2019). Centrosome loss triggers a transcriptional program to counter apoptosis-induced oxidative stress. Genetics. PubMed ID: 30867197
Centrosomes play a critical role in mitotic spindle assembly through their role in microtubule nucleation and bipolar spindle assembly. Loss of centrosomes can impair the ability of some cells to properly conduct mitotic division, leading to chromosomal instability, cell stress, and aneuploidy. Multiple aspects of the cellular response to mitotic error associated with centrosome loss appears to involve activation of JNK signaling. To further characterize the transcriptional effects of centrosome loss, gene expression profiles were compared of wildtype and acentrosomal cells from Drosophila wing imaginal discs. Elevation was found of expression of JNK target genes, which was verified at the protein level. Consistent with this, the upregulated gene set showed significant enrichment for the AP1 consensus DNA binding sequence. Significant elevation was found in expression of genes regulating redox balance. Based on those findings, oxidative stress after centrosome loss was examined, revealing that acentrosomal wing cells have significant increases in reactive oxygen species (ROS). A candidate genetic screen was performed, and one of the genes upregulated in acentrosomal cells, G6PD, was found to play an important role in buffering acentrosomal cells against increased ROS and helps protect those cells from cell death. These data and other recent studies have revealed a complex network of signaling pathways, transcriptional programs, and cellular processes that epithelial cells use to respond to stressors like mitotic errors to help limit cell damage and maintain normal tissue development.
Mao, D., Lin, G., Tepe, B., Zuo, Z., Tan, K. L., Senturk, M., Zhang, S., Arenkiel, B. R., Sardiello, M. and Bellen, H. J. (2019). VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway. Autophagy. PubMed ID: 30741620
Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. This study found that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. Loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. It is proposed that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation.
Xu, P., Damschroder, D., Zhang, M., Ryall, K. A., Adler, P. N., Saucerman, J. J., Wessells, R. J. and Yan, Z. (2018). Atg2, Atg9 and Atg18 in mitochondrial integrity, cardiac function and healthspan in Drosophila. J Mol Cell Cardiol 127: 116-124. PubMed ID: 30571977
In yeast, the Atg2-Atg18 complex regulates Atg9 recycling from phagophore assembly site during autophagy; their function in higher eukaryotes remains largely unknown. In a targeted screening in Drosophila melanogaster, this study shows that Mef2-GAL4-RNAi-mediated knockdown of Atg2, Atg9 or Atg18 in the heart and indirect flight muscles led to shortened healthspan (declined locomotive function) and lifespan. These flies displayed an accelerated age-dependent loss of cardiac function along with cardiac hypertrophy (increased heart tube wall thickness) and structural abnormality (distortion of the lumen surface). Using the Mef2-GAL4-MitoTimer mitochondrial reporter system and transmission electron microscopy, significant elongation of mitochondria and reduced number of lysosome-targeted autophagosomes containing mitochondria were observed in the heart tube but exaggerated mitochondrial fragmentation and reduced mitochondrial density in indirect flight muscles. These findings provide the first direct evidence of the importance of Atg2-Atg18/Atg9 autophagy complex in the maintenance of mitochondrial integrity and, regulation of heart and muscle functions in Drosophila, raising the possibility of augmenting Atg2-Atg18/Atg9 activity in promoting mitochondrial health and, muscle and heart function.

Tsogtbaatar, O., Won, J. H., Kim, G. W., Han, J. H., Bae, Y. K. and Cho, K. O. (2019). An ADAMTS Sol narae is required for cell survival in Drosophila. Sci Rep 9(1): 1270. PubMed ID: 30718556
Cell survival is essential for all living organisms to cope against multiple environmental insults. Intercellular signaling between dying and surviving cells plays an important role to ensure compensatory proliferation, preventing tissue loss after environmental stresses. This study shows that Sol narae (Sona), a Disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in Drosophila is required for cell survival. sona exhibited a positive genetic interaction with Death-associated inhibitor of apoptosis 1 (Diap1), and a negative genetic interaction with reaper (rpr). Transcription patterns of sona, Diap1, and rpr genes in the pouch region of wing discs were coordinately changed after irradiation. Interestingly, there was a negative correlation in the expression levels of Sona and DIAP1, and both cell types, one with high Sona level and the other with high Diap1 level, were resistant to irradiation-induced cell death. The sona-expressing cells rarely entered into cell cycle themselves but promoted the nearby cells to proliferate in irradiation conditions. These sona-expressing cells are able to upregulate Cyclin D (Cyc D) and increase tissue size. Furthermore, transient Sona overexpression increased survival rate and promoted development of flies in irradiation conditions. It is proposed that the two types of radiation-resistant cells, one with high Sona level and the other with high Diap1 level, communicate with dying cells and between each other for cell survival and proliferation in response to irradiation.
Cosolo, A., Jaiswal, J., Csordas, G., Grass, I., Uhlirova, M. and Classen, A. K. (2019). JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress. Elife 8. PubMed ID: 30735120
The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. This study demonstrates that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation.
Kurtz, P., Jones, A. E., Tiwari, B., Link, N., Wylie, A., Tracy, C., Kramer, H. and Abrams, J. M. (2019). Drosophila p53 directs non-apoptotic programs in postmitotic tissue. Mol Biol Cell: mbcE18120791. PubMed ID: 30892991
This study leveraged the Drosophila system to interrogate p53 function in a postmitotic context. In the developing embryo, p53 robustly activates important apoptotic genes in response to radiation-induced DNA damage. A p53 enhancer (p53RErpr) near the cell death gene reaper, forms chromatin contacts and enables p53 target activation across long genomic distances. Interestingly, this canonical p53 apoptotic program failed to activate in adult heads. Moreover, this failure to exhibit apoptotic responses was not associated with altered chromatin contacts. Instead, it was determined that p53 does not occupy the p53RErpr enhancer in this postmitotic tissue as it does in embryos. Through comparative RNA-seq and ChIP-seq studies of developing and postmitotic tissues, it was further determined that p53 regulates distinct transcriptional programs in adult heads, including DNA repair, metabolism and proteolysis genes. Strikingly, in the postmitotic context p53 binding landscapes were poorly correlated with nearby transcriptional effects, raising the possibility that p53 enhancers could be generally acting through long distances.

Tuesday, April 16th - Vesicles and the Synapse

Russo, A., Goel, P., Brace, E. J., Buser, C., Dickman, D. and DiAntonio, A. (2019). The E3 ligase Highwire promotes synaptic transmission by targeting the NAD-synthesizing enzyme dNmnat. EMBO Rep 20(3). PubMed ID: 30692130
The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila. Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD(+) biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading to a hypothesis that Highwire promotes evoked release by downregulating dNmnat. This study also shows that excess dNmnat is necessary in highwire mutants and sufficient in wild-type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD(+) synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD(+) biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse-inhibitory functions of dNmnat.
Bohme, M. A., McCarthy, A. W., Grasskamp, A. T., Beuschel, C. B., Goel, P., Jusyte, M., Laber, D., Huang, S., Rey, U., Petzoldt, A. G., Lehmann, M., Gottfert, F., Haghighi, P., Hell, S. W., Owald, D., Dickman, D., Sigrist, S. J. and Walter, A. M. (2019). Rapid active zone remodeling consolidates presynaptic potentiation. Nat Commun 10(1): 1085. PubMed ID: 30842428
Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) this study shows that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation.
Dason, J. S., Allen, A. M., Vasquez, O. E. and Sokolowski, M. B. (2019). Distinct functions of a cGMP-dependent protein kinase in nerve terminal growth and synaptic vesicle cycling. J Cell Sci. PubMed ID: 30837290
Sustained neurotransmission requires the tight coupling of synaptic vesicle (SV) exocytosis and endocytosis. The mechanisms underlying this coupling are poorly understood. This study tested the hypothesis that a cGMP-dependent protein kinase (PKG), encoded by the foraging (for) gene in Drosophila melanogaster, is critical for this process using a for null mutant, genomic rescues, and tissue specific rescues. This study uncoupled FOR's exocytic and endocytic functions in neurotransmission using a temperature-sensitive shibire mutant in conjunction with fluorescein-assisted light inactivation of FOR. A dual role was discovered for presynaptic FOR, where FOR inhibits SV exocytosis during low frequency stimulation by negatively regulating presynaptic Ca(2+) levels and maintains neurotransmission during high frequency stimulation by facilitating SV endocytosis. Additionally, glial FOR negatively regulated nerve terminal growth through TGF-beta signaling and this developmental effect was independent from FOR's effects on neurotransmission. Overall, FOR plays a critical role in coupling SV exocytosis and endocytosis, thereby balancing these two components to maintain sustained neurotransmission.
Driller, J. H., et al. (2019). Phosphorylation of the Bruchpilot N-terminus in Drosophila unlocks axonal transport of active zone building blocks. J Cell Sci 132(6). PubMed ID: 30745339
Protein scaffolds at presynaptic active zone membranes control information transfer at synapses. For scaffold biogenesis and maintenance, scaffold components must be safely transported along axons. A spectrum of kinases has been suggested to control transport of scaffold components, but direct kinase-substrate relationships and operational principles steering phosphorylation-dependent active zone protein transport are presently unknown. This study shows that extensive phosphorylation of a 150-residue unstructured region at the N-terminus of the highly elongated Bruchpilot (BRP) active zone protein is crucial for ordered active zone precursor transport in Drosophila. Point mutations that block SRPK79D kinase-mediated phosphorylation of the BRP N-terminus interfered with axonal transport, leading to BRP-positive axonal aggregates that also contain additional active zone scaffold proteins. Axonal aggregates formed only in the presence of non-phosphorylatable BRP isoforms containing the SRPK79D-targeted N-terminal stretch. It is assumeed that specific active zone proteins are pre-assembled in transport packages and are thus co-transported as functional scaffold building blocks. These results suggest that transient post-translational modification of a discrete unstructured domain of the master scaffold component BRP blocks oligomerization of these building blocks during their long-range transport.
Kramer, R., Rode, S. and Rumpf, S. (2019). Rab11 is required for neurite pruning and developmental membrane protein degradation in Drosophila sensory neurons. Dev Biol. PubMed ID: 30871987
Neurons, with their distinct neurites, require elaborate membrane trafficking pathways and regulation to uphold neurite identity and to be able to respond to neuronal or developmental stimuli. In a survey of trafficking regulators required for developmental dendrite pruning in Drosophila sensory neurons, the small GTPase Rab11, a regulator of recycling endosomes, was identified. Dendrite pruning requires the developmentally regulated degradation of the cell adhesion molecule Neuroglian, and loss of Rab11 causes defects in the developmental degradation of Neuroglian and another target, the ion channel Ppk26. Rab11 often links vesicles to molecular motors, and this study finds that loss of the microtubule motor dynein also leads to defective Neuroglian and Ppk26 degradation. Loss of Rab11 also leads to defects in larval dendrite elaboration, and Neuroglian and Ppk26 localization is already altered at this stage. These data highlight the importance of membrane protein recycling during development.
Patron, L. A., Nagatomo, K., Eves, D. T., Imad, M., Young, K., Torvund, M., Guo, X., Rogers, G. C. and Zinsmaier, K. E. (2019). Cul4 ubiquitin ligase cofactor DCAF12 promotes neurotransmitter release and homeostatic plasticity. J Cell Biol 218(3): 993-1010. PubMed ID: 30670470
This study genetically characterized the synaptic role of the Drosophila homologue of human DCAF12, a putative cofactor of Cullin4 (Cul4) ubiquitin ligase complexes. Deletion of Drosophila DCAF12 impairs larval locomotion and arrests development. At larval neuromuscular junctions (NMJs), DCAF12 is expressed presynaptically in synaptic boutons, axons, and nuclei of motor neurons. Postsynaptically, DCAF12 is expressed in muscle nuclei and facilitates Cul4-dependent ubiquitination. Genetic experiments identified several mechanistically independent functions of DCAF12 at larval NMJs. First, presynaptic DCAF12 promotes evoked neurotransmitter release. Second, postsynaptic DCAF12 negatively controls the synaptic levels of the glutamate receptor subunits GluRIIA, GluRIIC, and GluRIID. The down-regulation of synaptic GluRIIA subunits by nuclear DCAF12 requires Cul4. Third, presynaptic DCAF12 is required for the expression of synaptic homeostatic potentiation. It is suggested that DCAF12 and Cul4 are critical for normal synaptic function and plasticity at larval NMJs.

Monday, April 15th - Embryonic Development

Jin, S., O, J., Stellabotte, F., Brown, S. J. and Choe, C. P. (2019). Expression of teneurin-m/odd Oz during segmentation in the beetle Tribolium castaneum. Gene Expr Patterns 31: 26-31. PubMed ID: 30630105
The pair-rule gene teneurin-m/odd Oz (ten-m/odz) is required for the patterning of alternate segment boundaries in the early Drosophila embryo. Mutant phenotypes of ten-m/odz display a typical pair-rule phenotype in which odd-numbered segments are eliminated. Consistent with its pair-rule function, Ten-m/Odz protein is expressed in a seven-stripe pattern before the onset of gastrulation. While expression of ten-m/odz orthologues have been characterized in several vertebrate species, their expression patterns in non-Drosophila arthropods during embryonic segmentation have yet to be reported. This study has identified a Tribolium orthologue of ten-m/odz (Tc-ten-m/odz) and analyzed its expression patterns during embryonic segmentation. Tc-ten-m/odz expression was observed in a region of the growth zone, which appeared to be a potential mesodermal region, during germband elongation. Later, segmental expression appeared in the trunk after segments had already formed. In contrast to Drosophila, apparently Tc-ten-m/odz was neither expressed in the ectoderm of the growth zone where segmentation occurs, nor the ectoderm of trunk regions where segmentation is maintained. These findings suggest that Tc-ten-m/odz may not function as a pair rule gene in Tribolium segmentation.
Graham, P. L., Anderson, W. R., Brandt, E. A., Xiang, J. and Pick, L. (2019). Dynamic expression of Drosophila segmental cell surface-encoding genes and their pair-rule regulators. Dev Biol. PubMed ID: 30695684
Drosophila segmentation is regulated by a complex network of transcription factors that include products of the pair-rule genes (PRGs). PRGs are expressed in early embryos in the primorida of alternate segmental units, establishing the repeated, segmental body plan of the fly. Cell surface proteins containing Leucine rich repeats (LRR) play a variety of roles in development, and those expressed in segmental patterns likely impact segment morphogenesis. This study explored the relationships between the PRG network and segmentally expressed LRR-encoding (sLRR) genes. Expression of Toll2, Toll6, Toll7, Toll8 and tartan (trn) was examined in wild type or PRG mutant embryos. Expression of each sLRR-encoding gene is dynamic, but each has a unique register along the anterior-posterior axis. The registers for different sLRRs are off-set from one another resulting in a continually changing set of overlapping expression patterns among the sLRR-encoding genes themselves and between the sLRR-encoding genes and the PRGs. Accordingly, each sLRR-encoding gene is regulated by a unique combination of PRGs. These findings suggest that one role of the PRG network is to promote segmentation by establishing a cell surface code: each row of cells in the two-segment-wide primordia expresses a unique combination of sLRRs, thereby translating regulatory information from the PRGs to direct segment morphogenesis.
Ray, S., Rosenberg, M. I., Chanut-Delalande, H., Decaras, A., Schwertner, B., Toubiana, W., Auman, T., Schnellhammer, I., Teuscher, M., Valenti, P., Khila, A., Klingler, M. and Payre, F. (2019). The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning. Elife 8. PubMed ID: 30896406
Small open reading frames (smORFs) encoding 'micropeptides' exhibit remarkable evolutionary complexity. Conserved peptides encoded by mille-pattes (mlpt)/polished rice (pri)/tarsal less (tal) are essential for embryo segmentation in Tribolium but, in Drosophila, function in terminal epidermal differentiation and patterning of adult legs. This study shows that a molecular complex identified in Drosophila epidermal differentiation, comprising Mlpt peptides, ubiquitin-ligase Ubr3 and transcription factor Shavenbaby (Svb), represents an ancient developmental module required for early insect embryo patterning. Loss of segmentation function for this module in flies evolved concomitantly with restriction of Svb expression in early Drosophila embryos. Consistent with this observation, artificially restoring early Svb expression in flies causes segmentation defects that depend on mlpt function, demonstrating enduring potency of an ancestral developmental switch despite evolving embryonic patterning modes. These results highlight the evolutionary plasticity of conserved molecular complexes under the constraints of essential genetic networks.
Burguete, A. S., Francis, D., Rosa, J. and Ghabrial, A. (2019). The regulation of cell size and branch complexity in the terminal cells of the Drosophila tracheal system. Dev Biol. PubMed ID: 30735663
The terminal cells of the larval Drosophila tracheal system extend dozens of branched cellular processes, most of which become hollow intracellular tubes that support gas exchange with internal tissues. Previously, a forward genetic mosaic screen was undertaken to uncover the pathways regulating terminal cell size, morphogenesis, and the generation and maintenance of new intracellular tubes. This initial work identified several mutations affecting terminal cell size and branch number, and suggested that branch complexity and cell size are typically coupled but could be genetically separated. To deepen understanding of these processes, the molecular identities was undertaken of mutations in the genes sprout, denuded and asthmatic, that had been implicated in the initial screen. This paper reveals the molecular identity of these genes and describes their function in the context of the TOR and Hippo pathways, which are widely appreciated to be key regulators of cell and organ size.
Johnson, H. E. and Toettcher, J. E. (2019). Signaling dynamics control cell fate in the early Drosophila embryo. Dev Cell 48(3): 361-370.e363. PubMed ID: 30753836
The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? This study combined optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. Erk activity was found to be sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. The endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.
Petkova, M. D., Tkacik, G., Bialek, W., Wieschaus, E. F. and Gregor, T. (2019). Optimal decoding of cellular identities in a genetic network. Cell 176(4): 844-855.e815. PubMed ID: 30712870
In developing organisms, spatially prescribed cell identities are thought to be determined by the expression levels of multiple genes. Quantitative tests of this idea, however, require a theoretical framework capable of exposing the rules and precision of cell specification over developmental time. This study used the gap gene network in the early fly embryo as an example to show how expression levels of the four gap genes can be jointly decoded into an optimal specification of position with 1% accuracy. The decoder correctly predicts, with no free parameters, the dynamics of pair-rule expression patterns at different developmental time points and in various mutant backgrounds. Precise cellular identities are thus available at the earliest stages of development, contrasting the prevailing view of positional information being slowly refined across successive layers of the patterning network.These results suggest that developmental enhancers closely approximate a mathematically optimal decoding strategy.

Friday, March 12th - Disease Models

Das, S., Kumar, P., Verma, A., Maiti, T. K. and Mathew, S. J. (2019). Myosin heavy chain mutations that cause Freeman-Sheldon syndrome lead to muscle structural and functional defects in Drosophila. Dev Biol. PubMed ID: 30826400
Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. Transgenic Drosophila were generated expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and the effect of their expression on Drosophila muscle structure and function was characterized. The results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. Flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, these findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins.
Hope, K. A., McGinn, A. and Reiter, L. T. (2019). A genome-wide enhancer/suppressor screen for Dube3a interacting genes in Drosophila melanogaster. Sci Rep 9(1): 2382. PubMed ID: 30787400
The genetics underlying autism spectrum disorder (ASD) are complex. Approximately 3-5% of ASD cases arise from maternally inherited duplications of 15q11.2-q13.1, termed Duplication 15q syndrome (Dup15q). 15q11.2-q13.1 includes the gene UBE3A which is believed to underlie ASD observed in Dup15q syndrome. UBE3A is an E3 ubiquitin ligase that targets proteins for degradation and trafficking, so finding UBE3A substrates and interacting partners is critical to understanding Dup15q ASD. This study took an unbiased genetics approach to identify genes that genetically interact with Dube3a, the Drosophila melanogaster homolog of UBE3A. An enhancer/suppressor screen was conducted using a rough eye phenotype produced by Dube3a overexpression with GMR-GAL4. Using the DrosDel deficiency kit, 3 out of 346 deficiency lines were identified that enhanced rough eyes when crossed to two separate Dube3a overexpression lines, and subsequently IA2, GABA-B-R3, and lola were identified as single genes responsible for rough eye enhancement. Using the FlyLight GAL4 lines to express uas-Dube3a + uas-GFP in the endogenous lola pattern, an increase was observed in the GFP signal compared to uas-GFP alone, suggesting a transcriptional co-activation effect of Dube3a on the lola promoter region. These findings extend the role of Dube3a/UBE3A as a transcriptional co-activator, and reveal new Dube3a interacting genes.
Nixon, K. C. J., et al. (2019). A syndromic neurodevelopmental disorder caused by mutations in SMARCD1, a core SWI/SNF subunit needed for context-dependent neuronal gene regulation in flies. Am J Hum Genet. PubMed ID: 30879640
Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause neurodevelopmental disorders (NDDs). This study reports on five individuals with mutations in SMARCD1; the individuals present with developmental delay, intellectual disability, hypotonia, feeding difficulties, and small hands and feet. Trio exome sequencing proved the mutations to be de novo in four of the five individuals. Although the individuals presented in this study have dysmorphisms and some clinical overlap with these syndromes, they lack their typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, this study investigated the Drosophila ortholog Bap60 in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom-body-specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, this study identified an NDD caused by SMARCD1 mutations and establish a role for the SMARCD1 ortholog Bap60 in the regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development when neuronal circuits that are required for learning and memory are formed.
Nguyen, N. N., Rana, A., Goldman, C., Moore, R., Tai, J., Hong, Y., Shen, J., Walker, D. W. and Hur, J. H. (2019). Proteasome beta5 subunit overexpression improves proteostasis during aging and extends lifespan in Drosophila melanogaster. Sci Rep 9(1): 3170. PubMed ID: 30816680
The beta5 subunit of the proteasome has been shown in worms and in human cell lines to be regulatory. In these models, beta5 overexpression results in upregulation of the entire proteasome complex which is sufficient to increase proteotoxic stress resistance, improve metabolic parameters, and increase longevity. However, fundamental questions remain unanswered, including the temporal requirements for beta5 overexpression and whether beta5 overexpression can extend lifespan in other species. To determine if adult-only overexpression of the beta5 subunit can increase proteasome activity in a different model, this study characterized phenotypes associated with beta5 overexpression in Drosophila melanogaster adults. Adult-only overexpression of the beta5 subunit does not result in transcriptional upregulation of the other subunits of the proteasome as they do in nematodes and human cell culture. Despite this lack of a regulatory role, boosting beta5 expression increases the chymotrypsin-like activity associated with the proteasome, reduces both the size and number of ubiquitinated protein aggregates in aged flies, and increases longevity. Surprisingly, these phenotypes were not associated with increased resistance to acute proteotoxic insults or improved metabolic parameters.
Kadas, D., Papanikolopoulou, K., Xirou, S., Consoulas, C. and Skoulakis, E. M. C. (2019). Human Tau isoform-specific presynaptic deficits in a Drosophila central nervous system circuit. Neurobiol Dis 124: 311-321. PubMed ID: 30529489
Accumulation of normal or mutant human Tau isoforms in Central Nervous System (CNS) neurons of vertebrate and invertebrate models underlies pathologies ranging from behavioral deficits to neurodegeneration that broadly recapitulate human Tauopathies. Although some functional differences have begun to emerge, it is still largely unclear whether normal and mutant Tau isoforms induce differential effects on the synaptic physiology of CNS neurons. This study used the oligosynaptic Giant Fiber System in the adult Drosophila CNS to address this question and reveal that 3R and 4R isoforms affect distinct synaptic parameters. Whereas 0N3R increased failure rate upon high frequency stimulation, 0N4R compromised stimulus conduction and response speed at a specific cholinergic synapse in an age-dependent manner. In contrast, accumulation of the R406W mutant of 0N4R induced mild, age-dependent conduction velocity defects. Because 0N4R and its mutant isoform are expressed equivalently, this demonstrates that the defects are not merely consequent of exogenous human Tau accumulation and suggests distinct functional properties of 3R and 4R isoforms in cholinergic presynapses.
Mengel-From, J., Svane, A. M., Pertoldi, C., Kristensen, T. N., Loeschcke, V., Skytthe, A., Christensen, K., Lindahl-Jacobsen, R., Hjelmborg, J. and Christiansen, L. (2019). Advanced parental age at conception and sex affects mitochondrial DNA copy number in human and fruit flies. J Gerontol A Biol Sci Med Sci. PubMed ID: 30874797
Aging is a multifactorial trait caused by early as well as late life circumstances. A society trend that parents deliberately delay having children is of concern to health professionals, e.g. since advanced parental age at conception increases disease risk profiles in offspring. This work performed a study to see if advanced parental age at conception affects mitochondria DNA content, a cross species biomarker of general health, in adult human twin offspring and in a model organism. No deteriorated mitochondria DNA content was found at advanced parental age at conception, but human mitochondria DNA content was higher in females than males, and the difference was two fold higher at advanced maternal age at conception. Similar parental age effects and sex-specific differences in mitochondria DNA content were found in Drosophila melanogaster. In addition, parental longevity in humans associates with both mitochondria DNA content and parental age at conception. It is thus carefully proposed that a poorer disease risk profile from advanced parental age at conception might be surpassed by superior effects of parental successful late-life reproduction that associate with parental longevity.

Thursday, April 11th - Evolution

Castillo, D. M. and Moyle, L. C. (2019). Conspecific sperm precedence is reinforced, but postcopulatory sexual selection weakened, in sympatric populations of Drosophila. Proc Biol Sci 286(1899): 20182535. PubMed ID: 30900533
Sexual selection can accelerate speciation by driving the evolution of reproductive isolation, but forces driving speciation could also reciprocally feedback on sexual selection. This might be particularly important in the context of 'reinforcement', where selection acts directly to increase prezygotic barriers to reduce the cost of heterospecific matings. Using assays of sperm competition within and between two sister species, a signature of reinforcement is shown where these species interact: populations of Drosophila pseudoobscura that co-occur with sister species D. persimilis have an elevated ability to outcompete heterospecific sperm, consistent with selection for increased postcopulatory isolation. These D. pseudoobscura populations have decreased sperm competitive ability against conspecifics, reducing the opportunity for sexual selection within these populations. These findings demonstrate that direct selection to increase reproductive isolation against other species can compromise the efficacy of sexual selection within species, a collateral effect of reproductive traits responding to heterospecific interactions.
Hopkins, D. H., Rane, R. V., Younus, F., Coppin, C. W., Pandey, G., Jackson, C. J. and Oakeshott, J. G. (2019). The molecular basis for the neofunctionalization of the juvenile hormone esterase duplication in Drosophila. Insect Biochem Mol Biol 106: 10-18. PubMed ID: 30611903
The Drosophila melanogaster enzymes juvenile hormone esterase (DmJHE) and its duplicate, DmJHEdup, present ideal examples for studying the structural changes involved in the neofunctionalization of enzyme duplicates. DmJHE is a hormone esterase with precise regulation and highly specific activity for its substrate, juvenile hormone. DmJHEdup is an odorant degrading esterase (ODE) responsible for processing various kairomones in antennae. Phylogenetic analysis shows that the JHE lineage predates the hemi/holometabolan split. DmJHE has sufficient substrate promiscuity and activity against odorant esters for a duplicate to evolve a general ODE function against a range of mid-long chain food esters, as is shown in DmJHEdup. Both JHEs showed very similar active sites despite low sequence identity (30%). Both ODEs differed drastically from the JHEs and each other, explaining their complementary substrate ranges. A small number of amino acid changes are identified that may have been involved in the early stages of the neofunctionalization of DmJHEdup. These results provide key insights into the process of neofunctionalization and the structural changes that can be involved.
Nguyen, T. T. X. and Moehring, A. J. (2019). Males from populations with higher competitive mating success produce sons with lower fitness. J Evol Biol. PubMed ID: 30811733
Female mate choice can result in direct benefits to the female or indirect benefits through her offspring. Females can increase their fitness by mating with males whose genes encode increased survivorship and reproductive output. Alternatively, male investment in enhanced mating success may come at the cost of reduced investment in offspring fitness. This study measures male mating success in a mating arena that allows for male-male, male-female and female-female interactions in Drosophila melanogaster. Isofemale line population measurements were used to correlate male mating success with sperm competitive ability, the number of offspring produced and the indirect benefits of the number of offspring produced by daughters and sons. This study finds that males from populations that gain more copulations do not increase female fitness through increased offspring production, nor do these males fare better in sperm competition. Instead, it was found that these populations have a reduced reproductive output of sons, indicating a potential reproductive trade-off between male mating success and offspring quality.
Keesey, I. W., Grabe, V., Gruber, L., Koerte, S., Obiero, G. F., Bolton, G., Khallaf, M. A., Kunert, G., Lavista-Llanos, S., Valenzano, D. R., Rybak, J., Barrett, B. A., Knaden, M. and Hansson, B. S. (2019). Inverse resource allocation between vision and olfaction across the genus Drosophila. Nat Commun 10(1): 1162. PubMed ID: 30858374
Divergent populations across different environments are exposed to critical sensory information related to locating a host or mate, as well as avoiding predators and pathogens. These sensory signals generate evolutionary changes in neuroanatomy and behavior; however, few studies have investigated patterns of neural architecture that occur between sensory systems, or that occur within large groups of closely-related organisms. This study examine 62 species within the genus Drosophila and describes an inverse resource allocation between vision and olfaction, which was consistently observe at the periphery, within the brain, as well as during larval development. This sensory variation was noted across the entire genus and appears to represent repeated, independent evolutionary events, where one sensory modality is consistently selected for at the expense of the other. Moreover, evidence is provided of a developmental genetic constraint through the sharing of a single larval structure, the eye-antennal imaginal disc. In addition, the ecological implications of visual or olfactory bias were examined, including the potential impact on host-navigation and courtship.
Ding, Y., Lillvis, J. L., Cande, J., Berman, G. J., Arthur, B. J., Long, X., Xu, M., Dickson, B. J. and Stern, D. L. (2019). Neural evolution of context-dependent fly song. Curr Biol. PubMed ID: 30880014
It is unclear where in the nervous system evolutionary changes tend to occur. To localize the source of neural evolution that has generated divergent behaviors, a new approach was developed to label and functionally manipulate homologous neurons across Drosophila species. Homologous descending neurons were examined that drive courtship song in two species that sing divergent song types, and relevant evolutionary changes were localized in circuit function downstream of the intrinsic physiology of these descending neurons. This evolutionary change causes different species to produce divergent motor patterns in similar social contexts. Artificial stimulation of these descending neurons drives multiple song types, suggesting that multifunctional properties of song circuits may facilitate rapid evolution of song types.
Bernardo-Garcia, F. J., Syed, M., Jekely, G. and Sprecher, S. G. (2019). Glass confers rhabdomeric photoreceptor identity in Drosophila, but not across all metazoans. Evodevo 10: 4. PubMed ID: 30873275
Across metazoans, visual systems employ different types of photoreceptor neurons (PRs) to detect light. These include rhabdomeric PRs, which exist in distantly related phyla and possess an evolutionarily conserved phototransduction cascade. Very little is known about how rhabdomeric PRs form in species other than Drosophila. To investigate this question, tests were performed to see whether the transcription factor Glass, which is crucial for instructing rhabdomeric PR formation in Drosophila, may play a similar role in other metazoans. Glass homologues exist throughout the animal kingdom, indicating that this protein evolved prior to the metazoan radiation. Interestingly, this work indicates that glass is not expressed in rhabdomeric photoreceptors in the planarian Schmidtea mediterranea nor in the annelid Platynereis dumerilii. Combined with a comparative analysis of the Glass DNA-binding domain, these data suggest that the fate of rhabdomeric PRs is controlled by Glass-dependent and Glass-independent mechanisms in different animal clades.

Wednesday, April 10th - Signaling

Nagaoka, T., Furuse, M., Ohtsuka, T., Tsuchida, K. and Kishi, M. (2019). Vangl2 interaction plays a role in the proteasomal degradation of Prickle2. Sci Rep 9(1): 2912. PubMed ID: 30814664
The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, the regulation of Prickle2 protein levels was studied in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2; see Drosophila Van Gogh). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.
Kim, A. R. and Choi, K. W. (2019). TRiC/CCT chaperonins are essential for organ growth by interacting with insulin/TOR signaling in Drosophila. Oncogene. PubMed ID: 30792539
Organ size is regulated by intercellular signaling for cell growth and proliferation. The TOR pathway mediates a key signaling mechanism for controlling cell size and number in organ growth. Chaperonin containing TCP-1 (CCT) is a complex that assists protein folding and function, but its role in animal development is largely unknown. This study shows that the CCT complex is required for organ growth by interacting with the TOR pathway in Drosophila. Reduction of CCT4 results in growth defects by affecting both cell size and proliferation. Loss of CCT4 causes preferential cell death anterior to the morphogenetic furrow in the eye disc and within wing pouch in the wing disc. Depletion of any CCT subunit in the eye disc results in headless phenotype. Overgrowth by active TOR signaling is suppressed by CCT RNAi. The CCT complex physically interacts with TOR signaling components including TOR, Rheb, and S6K. Loss of CCT leads to decreased phosphorylation of S6K and S6 while increasing phosphorylation of Akt. Insulin/TOR signaling is also necessary and sufficient for promoting CCT complex transcription. These data provide evidence that the CCT complex regulates organ growth by directly interacting with the TOR signaling pathway.
Loza-Coll, M. A., Petrossian, C. C., Boyle, M. L. and Jones, D. L. (2019). Heterochromatin Protein 1 (HP1) inhibits stem cell proliferation induced by ectopic activation of the Jak/STAT pathway in the Drosophila testis. Exp Cell Res 377(1-2): 1-9. PubMed ID: 30817931
Stem cells can divide asymmetrically with respect to cell fate, producing a copy of themselves (self-renewal), while giving rise to progeny that will differentiate along a specific lineage. Mechanisms that bias the balance towards self-renewal or extend the proliferative capacity of the differentiating progeny can result in tissue overgrowth and, eventually, the formation of tumors. Recent work has explored the role of heterochromatin and heterochromatin-associated proteins in the regulation of stem cell behavior under homeostatic conditions, but less is known about their possible roles in potentiating or suppressing stem cell overproliferation. This studyused ectopic activation of the Jak/STAT pathway in germline and somatic stem cells of the D. melanogaster testis as an in vivo model to probe the function of Heterochromatin Protein 1 (HP1) in stem cell overproliferation. Forced expression of HP1 in either early germ or somatic cells suppressed the overgrowth of testes in response to ectopic Jak/STAT activation. Interestingly, HP1 expression led to distinct phenotypes, depending on whether it was overexpressed in somatic or germ cells, possibly reflecting different cell-autonomous and non-autonomous effects in each cell type. These results provide a new framework for further in vivo studies aimed at understanding the interactions between heterochromatin and uncontrolled stem cell proliferation, as well as the complex cross-regulatory interactions between the somatic and germline lineages in the Drosophila testis.
Maiti, S., Acharya, B., Boorla, V. S., Manna, B., Ghosh, A. and De, S. (2019). Dynamic studies on intrinsically disordered regions of two paralogous transcription factors reveal rigid segments with important biological functions. J Mol Biol. PubMed ID: 30802457
Long stretches of intrinsically disordered regions (IDRs) are abundantly present in eukaryotic transcription factors. Although their biological significance is well appreciated, the underlying structural and dynamic mechanisms of their function are still not clear. Using solution NMR spectroscopy, the structural and dynamic features of two paralogous HOX transcription factors, SCR and DFD, from Drosophila were studied. Both proteins have a conserved DNA-binding homeodomain and a long stretch of functionally important IDR. Using NMR dynamics, flexibility of each residue in these proteins was determined. The flexibility of the residues in the disordered region is not uniform. In both proteins, the IDRs have short stretches of consecutive residues with relatively less flexibility, that is, higher rigidity. One such rigid segment is specifically recognized by another co-transcription factor, thus highlighting the importance of these rigid segments in IDR-mediated protein-protein interactions. Using molecular dynamics simulation, it was further showm that the rigid segments sample less conformations compared to the rest of the residues in the disordered region. The restrained conformational sampling of these rigid residues should lower the loss in conformational entropy during their interactions with binding partners resulting in sequence specific binding. This work provides experimental evidence of a "rigid-segment" model of IDRs, where functionally important rigid segments are connected by highly flexible linkers. Furthermore, a comparative study of IDRs in paralogous proteins reveals that in spite of low-sequence conservation, the rigid and flexible segments are sequentially maintained to preserve related functions and regulations of these proteins.
McSharry, S. S. and Beitel, G. J. (2019). The Caspase-3 homolog DrICE regulates endocytic trafficking during Drosophila tracheal morphogenesis. Nat Commun 10(1): 1031. PubMed ID: 30833576
Although well known for its role in apoptosis, the executioner caspase DrICE has a non-apoptotic function that is required for elongation of the epithelial tubes of the Drosophila tracheal system. This study shows that DrICE acts downstream of the Hippo Network to regulate endocytic trafficking of at least four cell polarity, cell junction and apical extracellular matrix proteins involved in tracheal tube size control: Crumbs, Uninflatable, Kune-Kune and Serpentine. Tracheal cells are competent to undergo apoptosis, even though developmentally-regulated DrICE function rarely kills tracheal cells. The results reveal a developmental role for caspases, a pool of DrICE that co-localizes with Clathrin, and a mechanism by which the Hippo Network controls endocytic trafficking. Given reports of in vitro regulation of endocytosis by mammalian caspases during apoptosis, it is proposed that caspase-mediated regulation of endocytic trafficking is an evolutionarily conserved function of caspases that can be deployed during morphogenesis.
Moskalev, A., Proshkina, E., Zhavoronkov, A. and Shaposhnikov, M. (2019). Effects of unpaired 1 gene overexpression on the lifespan of Drosophila melanogaster. BMC Syst Biol 13(Suppl 1): 16. PubMed ID: 30836998
The JAK/STAT signaling pathway is involved in many aging-related cellular functions. However, effects of overexpression of genes controlling JAK/STAT signal transduction on longevity of model organisms have not been studied. This study evaluate the effect of overexpression of the unpaired 1 (upd1) gene, which encodes an activating ligand for JAK/STAT pathway, on the lifespan of Drosophila melanogaster. Overexpression of upd1 in the intestine caused a pronounced shortening of the median lifespan by 54.1-18.9%, and the age of 90% mortality by 40.9-19.1% in males and females, respectively. In fat body and in nervous system of male flies, induction of upd1 overexpression increased the age of 90% mortality and median lifespan, respectively. An increase in upd1 expression enhanced mRNA levels of the JAK/STAT target genes domeless and Socs36E. It is concluded that conditional overexpression of upd1 in different tissues of Drosophila imago induces pro-aging or pro-longevity effects in tissue-dependent manner. The effects of upd1 overexpression on lifespan are accompanied by the transcription activation of genes for the components of JAK/STAT pathway.

Tuesday, April 9th - RNA

Kotov, A. A., Adashev, V. E., Godneeva, B. K., Ninova, M., Shatskikh, A. S., Bazylev, S. S., Aravin, A. A. and Olenina, L. V. (2019). piRNA silencing contributes to interspecies hybrid sterility and reproductive isolation in Drosophila melanogaster. Nucleic Acids Res. PubMed ID: 30788506
The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Suppressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, it was discovered that AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.
Li, J., Yu, R. Y., Emran, F., Chen, B. E. and Hughes, M. E. (2019). Achilles-mediated and sex-specific regulation of circadian mRNA rhythms in Drosophila. J Biol Rhythms: 748730419830845. PubMed ID: 30803307
The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. This study explored the role of Achilles in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain, and legacy data suggest it is not expressed in core clock neurons. To assess its impact on circadian mRNA rhythms, RNA sequencing (RNAseq) was performed to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Dramatic upregulation of immune response genes was observed upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.
Grima, B., Papin, C., Martin, B., Chelot, E., Ponien, P., Jacquet, E. and Rouyer, F. (2019). Period-controlled deadenylation of the timeless transcript in the Drosophila circadian clock. Proc Natl Acad Sci U S A 116(12): 5721-5726. PubMed ID: 30833404
The Drosophila circadian oscillator relies on a negative transcriptional feedback loop, in which the Period (Per) and Timeless (Tim) proteins repress the expression of their own gene by inhibiting the activity of the Clock (Clk) and Cycle (Cyc) transcription factors. A series of posttranslational modifications contribute to the oscillations of the Per and Tim proteins but few posttranscriptional mechanisms have been described that affect mRNA stability. This study reports that down-regulation of the POP2 deadenylase, a key component of the CCR4-NOT deadenylation complex, alters behavioral rhythms. Down-regulating POP2 specifically increases Tim protein and tim mRNA but not tim pre-mRNA, supporting a posttranscriptional role. Indeed, reduced POP2 levels induce a lengthening of tim mRNA poly(A) tail. Surprisingly, such effects are lost in per (0) mutants, supporting a Per-dependent inhibition of tim mRNA deadenylation by POP2. This study reports a deadenylation mechanism that controls the oscillations of a core clock gene transcript.
Hanyu-Nakamura, K., Matsuda, K., Cohen, S. M. and Nakamura, A. (2019). Pgc suppresses the zygotically-acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo. Development. PubMed ID: 30890569
Specification of germ cells is pivotal to ensure continuation of animal species. In many animal embryos, germ cell specification depends on maternally supplied determinants in the germ plasm. Drosophila polar granule component (pgc) mRNA is a component of the germ plasm. pgc encodes a small protein that is transiently expressed in newly formed pole cells, the germline progenitors, where it globally represses mRNA transcription. pgc is also required for pole cell survival, but the mechanism linking transcriptional repression to pole cell survival remains elusive. This study reports that pole cells lacking pgc show premature loss of germ plasm mRNAs, including the germ cell survival factor, nanos, and undergo apoptosis. pgc (-) pole cells misexpress multiple miRNA genes. Reduction of miRNA pathway activity in pgc (-) embryos partially suppressed germ plasm mRNA degradation and pole cell death, suggesting that Pgc represses zygotic miRNA transcription in pole cells to protect germ plasm mRNAs. Interestingly, germ plasm mRNAs are protected from miRNA-mediated degradation in vertebrates, albeit by a different mechanism. Thus, independently evolved mechanisms are used to silence miRNAs during germ cell specification.
Ji, S., Luo, Y., Cai, Q., Cao, Z., Zhao, Y., Mei, J., Li, C., Xia, P., Xie, Z., Xia, Z., Zhang, J., Sun, Q. and Chen, D. (2019). LC domain-mediated coalescence is essential for Otu enzymatic activity to extend Drosophila lifespan. Mol Cell. PubMed ID: 30879902
In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. This study shows that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.
Lee, M., Nguyen, T. M. T. and Kim, K. (2019). In-depth study of lin-28 suggests selectively conserved let-7 independent mechanism in Drosophila. Gene 687: 64-72. PubMed ID: 30415010
Lin-28 is a conserved RNA-binding protein that is involved in a wide range of developmental processes and pathogenesis. At the molecular level, Lin-28 blocks the maturation of let-7 and regulates translation of certain mRNA targets. In Drosophila, Lin-28 is reported to play a role in oogenesis, muscle formation, and the symmetric division of adult intestinal stem cells. This study characterized Drosophila Lin-28 through a detailed examination of its temporal and spatial expression. Lin-28 is specifically expressed in embryonic nervous and cardiac systems. However, loss or gain of lin-28 function does not cause any abnormality during embryonic development. Instead, the ubiquitous overexpression of Lin-28 leads to lethality from late larval stage to pupal stage, and eye-specific overexpression causes severe cell loss. The ectopic expression of human Lin28A has the same effect as Drosophila Lin-28, indicating functional conservation in Lin-28 orthologs. The effect of Lin-28 on let-7 biogenesis was also studied through the mutant and overexpression analysis. Lin-28 does not block the production of let-7 in Drosophila, which suggests the let-7 independent pathway as a molecular mechanism of Lin-28.

Monday, April 8th - Chromatin and Transposons

Soffers, J. H. M., Li, X., Saraf, A., Seidel, C. W., Florens, L., Washburn, M. P., Abmayr, S. M. and Workman, J. L. (2019). Characterization of a metazoan ADA acetyltransferase complex. Nucleic Acids Res. PubMed ID: 30715476
The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been described in metazoans. Previous studies in Drosophila hinted at the existence of a small complex which contains Ada2b, a partner of Gcn5 in the SAGA complex. This study has purified and characterized the composition of this complex and shows that it is composed of Gcn5, Ada2b, Ada3 and Sgf29. Hence, it was named the metazoan 'ADA complex'. The fly ADA complex has histone acetylation activity on histones and nucleosome substrates. Moreover, ChIP-Sequencing experiments identified Ada2b peaks that overlap with another SAGA subunit, Spt3, as well as Ada2b peaks that do not overlap with Spt3 suggesting that the ADA complex binds chromosomal sites independent of the larger SAGA complex.
Rass, M., Oestreich, S., Guetter, S., Fischer, S. and Schneuwly, S. (2019). The Drosophila fussel gene is required for bitter gustatory neuron differentiation acting within an Rpd3 dependent chromatin modifying complex. PLoS Genet 15(2): e1007940. PubMed ID: 30730884
Members of the Ski/Sno protein family are classified as proto-oncogenes and act as negative regulators of the TGF-β/BMP-pathways. A newly identified member of this protein family is fussel (fuss), the Drosophila homologue of the human functional Smad suppressing elements (fussel-15 and fussel-18). Fuss interacts with SMAD4 and overexpression leads to a strong inhibition of Dpp signaling. Fuss is a predominantly nuclear, postmitotic protein, mainly expressed in interneurons and fuss mutants are fully viable without any obvious developmental phenotype. fuss expression was characterized in the adult proboscis, and by using food choice assays it was possible to show that fuss mutants display defects in detecting bitter compounds. This correlated with a reduction of gustatory receptor gene expression providing a molecular link to the behavioral phenotype. In addition, Fuss interacts with Rpd3, and downregulation of rpd3 in gustatory neurons phenocopies the loss of Fuss expression. Surprisingly, there is no colocalization of Fuss with phosphorylated Mad in the larval central nervous system, excluding a direct involvement of Fuss in Dpp/BMP signaling. This work reveals Fuss as a pivotal element for the proper differentiation of bitter gustatory neurons acting within a chromatin modifying complex.
Jagannathan, M., Cummings, R. and Yamashita, Y. M. (2019). The modular mechanism of chromocenter formation in Drosophila. Elife 8. PubMed ID: 30741633
A central principle underlying the ubiquity and abundance of pericentromeric satellite DNA repeats in eukaryotes has remained poorly understood. It has been proposed that the interchromosomal clustering of satellite DNAs into nuclear structures known as chromocenters ensures encapsulation of all chromosomes into a single nucleus. Chromocenter disruption led to micronuclei formation, resulting in cell death. This study shows that chromocenter formation is mediated by a 'modular' network, where associations between two sequence-specific satellite DNA-binding proteins, D1 and Prod, bound to their cognate satellite DNAs, bring the full complement of chromosomes into the chromocenter. D1 prod double mutants die during embryogenesis, exhibiting enhanced phenotypes associated with chromocenter disruption, revealing the universal importance of satellite DNAs and chromocenters. Taken together, it is proposed that associations between chromocenter modules, consisting of satellite DNA binding proteins and their cognate satellite DNA, package the Drosophila genome within a single nucleus.
Carlson, J., Swisse, T., Smith, C. and Deng, H. (2019). Regulation of position effect variegation at pericentric heterochromatin by Drosophila Keap1-Nrf2 xenobiotic response factors. Genesis: e23290. PubMed ID: 30888733
The Keap1-Nrf2 signaling pathway plays a central role in the regulation of transcriptional responses to oxidative species and xenobiotic stimuli. The complete range of molecular mechanisms and biological functions of Keap1 and Nrf2 remain to be fully elucidated. To determine the potential roles of Keap1 and Nrf2 in chromatin architecture, the effects of their Drosophila homologs (dKeap1 and CncC) were examined on position effect variegation (PEV), which is a transcriptional reporter for heterochromatin formation and spreading. Loss of function mutations in cncC, dKeap1, and cncC/dKeap1 double mutants all suppressed the variegation of w(m4) and Sb(V) PEV alleles, indicating that reduction of CncC or dKeap1 causes a decrease of heterochromatic silencing at pericentric region. Depletion of CncC or dKeap1 in embryos reduced the level of the H3K9me2 heterochromatin marker, but had no effect on the transcription of the genes encoding Su(var)3-9 and HP1. These results support a potential role of dKeap1 and CncC in the establishment and/or maintenance of pericentric heterochromatin. This study provides preliminary evidence for a novel epigenetic function of Keap1-Nrf2 oxidative/xenobiotic response factors in chromatin remodeling.
Dorafshan, E., Kahn, T. G., Glotov, A., Savitsky, M., Walther, M., Reuter, G. and Schwartz, Y. B. (2019). Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation. EMBO Rep. PubMed ID: 30833342
Polycomb repression is critical for metazoan development. Equally important but less studied is the Trithorax system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. It was proposed that the Trithorax system acts via methylation of histone H3 at lysine 4 and lysine 36 (H3K36), thereby inhibiting histone methyltransferase activity of the Polycomb complexes. This hypothesis was tested by asking whether the Trithorax group protein Ash1 requires H3K36 methylation to counteract Polycomb repression. This study shows that Ash1 is the only Drosophila H3K36-specific methyltransferase necessary to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, the experiments reveal no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, it was found that complete substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not cause excessive repression of homeotic genes. These results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, needs revision.
Lam, K. C., Chung, H. R., Semplicio, G., Iyer, S. S., Gaub, A., Bhardwaj, V., Holz, H., Georgiev, P. and Akhtar, A. (2019). The NSL complex-mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise. Genes Dev. PubMed ID: 30819819
Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDRs and the precise positioning of the +1 nucleosomes are maintained on active genes remains unclear. This study reports that the Drosophila nonspecific lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. Not only is the NSL complex essential for transcription, but it is required for accurate TSS selection for genes with multiple TSSs. Furthermore, loss of the NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

Friday April 5th - Cytoskeleton and Junctions

Kuo, Y. W., Trottier, O., Mahamdeh, M. and Howard, J. (2019). Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules. Proc Natl Acad Sci U S A 116(12): 5533-5541. PubMed ID: 30837315
The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases-spastin, katanin, and fidgetin-which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, this study reconstituted Drosophila spastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. It was observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin's effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin's in vivo phenotypes.
Harker, A. J., Katkar, H. H., Bidone, T. C., Aydin, F., Voth, G. A., Applewhite, D. A. and Kovar, D. R. (2019). Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin. Mol Biol Cell 30(7): 851-862. PubMed ID: 30601697
Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (alpha-actinin). Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. This study used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP's processive mechanism on bundled filaments. Ena's enhanced processivity on trailing barbed ends was found to be specific to fascin bundles, with no enhancement on fimbrin or alpha-actinin bundles. Notably, Ena/VASP's processive run length increases with the number of both fascin-bundled filaments and Ena "arms," revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, this study discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.
Casas-Tinto, S. and Ferrus, A. (2019). Troponin-I localizes selected apico-basal cell polarity signals. J Cell Sci. PubMed ID: 30872455
Beyond its role in muscle contraction, Drosophila Troponin I (TnI) is expressed in epithelial cells where it controls proliferation. TnI traffics between nucleus and cytoplasm through a sumoylation-dependent mechanism. This study addresses the TnI role in the cytoplasm. TnI accumulates apically in epidermal cells and neuroblasts. TnI co-immunoprecipitates with Par-3/Bazooka and Disc large (Dlg), two apico-basal polarity components. By contrast, Scribbled is not altered by TnI depletion. In neuroblasts, TnI contributes to the polar localization of Miranda while non-polar Dlg is not affected. Vertebrate PI3K contributes to apico-basal polarity of epithelia but Drosophila PI3K depletion alters neither apical TnI or Par3/Bazooka, nor basal Dlg. Nevertheless, overexpressing PI3K prevents TnI depletion defects. TnI loss-of-function disrupts cytoskeletal beta-Catenin, E-Cadherin and gamma-Tubulin localization, along with gammaH2Av revealed DNA damage. The TnI-dependent apoptosis is suppressible upregulating Sparc or downregulating Dronc. Rescue from apoptosis by p35 does not prevent DNA damage demonstrating that both features are mechanistically independent. Thus, TnI binds certain apico-basal polarity signals in a cell type dependent context, and it unveils a hereto unsuspected diversity of mechanisms to allocate cell polarity factors.
Swider, Z. T., Ng, R. K., Varadarajan, R., Fagerstrom, C. J. and Rusan, N. M. (2019). Fascetto (PRC1) interacting protein (FIP) ensures proper cytokinesis and ploidy. Mol Biol Cell: mbcE18090573. PubMed ID: 30726162
Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring, and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is Protein Regulator of Cytokinesis 1 (PRC1), or Fascetto (in Drosophila, which forms a homodimer to crosslink interzonal MTs, ensuring proper central spindle formation and cytokinesis. This study reports on a new direct protein interactor and regulator of Feo named Fascetto Interacting Protein (FIP; Sip2). Loss of FIP results in a reduction in Feo localization, rapid disassembly of interzonal MTs, and several defects related to cytokinesis failure, include polyploidization of neural stem cells. Simultaneous reduction in Feo and FIP results in very large, tumor-like, DNA-filled masses in the brain that contain hundreds of centrosomes. In aggregate, these data show that FIP acts directly on Feo to ensure fully accurate cell division.
Khadilkar, R. J. and Tanentzapf, G. (2019). Septate junction components control Drosophila hematopoiesis through the Hippo pathway. Development. PubMed ID: 30890573
Hematopoiesis requires co-ordinated cell signals to control the proliferation and differentiation of progenitor cells. In Drosophila, blood progenitors, called prohemocytes, located in a hematopoietic organ called the lymph gland, are regulated by the Salvador-Warts-Hippo pathway. In epithelial cells the Hippo pathway integrates diverse biological inputs such as cell polarity and cell-cell contacts, but Drosophila blood cells lack the conspicuous polarity of epithelial cells. This study shows that the septate-junction components Cora and NrxIV promote Hippo signalling in the lymph gland. Depletion of septate-junction components in hemocytes produces similar phenotypes to those observed in Hippo pathway mutants including increased differentiation of immune cells. This analysis places septate-junction components as upstream regulators of the Hippo pathway where they recruit Merlin and Expanded to the membrane. Finally, this study shows that interactions of septate-junction components with the Hippo pathway are a key functional component of the cellular immune response following infection.
Verma, V. and Maresca, T. J. (2019). Microtubule plus-ends act as physical signaling hubs to activate RhoA during cytokinesis. Elife 8. PubMed ID: 30758285
Microtubules (MTs) are essential for cleavage furrow positioning during cytokinesis, but the mechanisms by which MT-derived signals spatially define regions of cortical contractility are unresolved. In this study cytokinesis regulators visualized in Drosophila melanogaster (Dm) cells were found to localize to and track MT plus-ends during cytokinesis. The RhoA GEF ECT2) did not evidently tip-track, but rather localized rapidly to cortical sites contacted by MT plus-tips, resulting in RhoA activation and enrichment of myosin-regulatory light chain. The MT plus-end localization of centralspindlin was compromised following EB1 depletion, which resulted in a higher incidence of cytokinesis failure. Centralspindlin plus-tip localization depended on the C-terminus and a putative EB1-interaction motif (hxxPTxh) in RacGAP50C. It is proposed that MT plus-end-associated centralspindlin recruits a cortical pool of Dm ECT2 upon physical contact to activate RhoA and to trigger localized contractility.

Thursday, March 4th - Adult Physiology

Scarpati, M., Qi, Y., Govind, S. and Singh, S. (2019). A combined computational strategy of sequence and structural analysis predicts the existence of a functional eicosanoid pathway in Drosophila melanogaster. PLoS One 14(2): e0211897. PubMed ID: 30753230
This study reports on a putative eicosanoid biosynthesis pathway in Drosophila melanogaster and challenges the currently held view that mechanistic routes to synthesize eicosanoid or eicosanoid-like biolipids do not exist in insects. Systematic and comprehensive bioinformatics approaches were used to identify most of the mammalian eicosanoid synthesis enzymes. Sensitive sequence analysis techniques identified candidate Drosophila enzymes that share low global sequence identities with their human counterparts. Twenty Drosophila candidates were selected based upon (a) sequence identity with human enzymes of the cyclooxygenase and lipoxygenase branches, (b) similar domain architecture and structural conservation of the catalytic domain, and (c) presence of potentially equivalent functional residues. Evaluation of full-length structural models for these 20 top-scoring Drosophila candidates revealed a surprising degree of conservation in their overall folds and potential analogs for functional residues in all 20 enzymes. Although a suitable candidate for lipoxygenase enzymes was not identified, this study reports structural homology models of three fly cyclooxygenases. These findings predict that the D. melanogaster genome likely codes for one or more pathways for eicosanoid or eicosanoid-like biolipid synthesis. This study suggests that classical and/or novel eicosanoids mediators must regulate biological functions in insects-predictions that can be tested with the power of Drosophila genetics.
Cormier, R. P. J., Champigny, C. M., Simard, C. J., St-Coeur, P. D. and Pichaud, N. (2019). Dynamic mitochondrial responses to a high-fat diet in Drosophila melanogaster. Sci Rep 9(1): 4531. PubMed ID: 30872605
Mitochondria can utilize different fuels according to physiological and nutritional conditions to promote cellular homeostasis. However, during nutrient overload metabolic inflexibility can occur, resulting in mitochondrial dysfunctions. High-fat diets (HFDs) are usually used to mimic this metabolic inflexibility in different animal models. However, how mitochondria respond to the duration of a HFD exposure is still under debate. This study investigated the dynamic of the mitochondrial and physiological functions in Drosophila melanogaster at several time points following an exposure to a HFD. The results showed that after two days on the HFD, mitochondrial respiration as well as ATP content of thorax muscles are increased, likely due to the utilization of carbohydrates. However, after four days on the HFD, impairment of mitochondrial respiration at the level of complex I, as well as decreased ATP content were observed. This was associated with an increased contribution of complex II and, most notably of the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH) to mitochondrial respiration. It is suggested that this increased mG3PDH capacity reflects the occurrence of metabolic inflexibility, leading to a loss of homeostasis and alteration of the cellular redox status, which results in senescence characterized by decreased climbing ability and premature death.
Kim, J. H., Moreau, J. A., Zina, J. M., Mazgaeen, L., Yoon, K. S., Pittendrigh, B. R. and Clark, J. M. (2018). Identification and interaction of multiple genes resulting in DDT resistance in the 91-R strain of Drosophila melanogaster by RNAi approaches. Pestic Biochem Physiol 151: 90-99. PubMed ID: 30704719
DDT has been re-recommended by the WHO for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will likely increase in terms of level and extent. Drosophila is a model dipteran that allows both forward and reverse genetic manipulation, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold) and recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms. Their interactions, however, remain unclear. Use of Gal4/UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has identified the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in decreased sensitivity to DDT. These above findings have been further validated in 91-R flies using a nanoparticle-enhanced RNAi strategy, directly implication these genes in DDT resistance in 91-R flies.
Inomata, N., Takahasi, K. R. and Koga, N. (2019). Association between duplicated maltase genes and the transcriptional regulation for the carbohydrate changes in Drosophila melanogaster. Gene 686: 141-145. PubMed ID: 30399425
Gene duplication could promote phenotypic and genetic adaptation to various environments. To understand the effects of gene duplication on transcriptional regulation associated with environmental changes, this study focused on the starch hydrolysis pathway, in which amylase enzymes together with maltase enzymes hydrolyze starch into glucose. Drosophila genomes involve ten duplicated Maltase genes. The levels of transcription of the nine of these genes was examined in 36 lines of Drosophila melanogaster collected from a natural population. In the investigated population, the levels of transcription were different between the two dietary carbohydrate sources, glucose and starch. At the transcriptional level, a single Maltase gene, which transcribes the specific Maltase transcripts, worked together with an Amylase gene in the pathway. The three of nine genes responded to carbohydrate changes, and the degree of the response was similar to Amylase gene. These results suggest that gene duplication could increase capacity of the transcriptional regulation associated with environmental changes.
Justice, A. E., et al. (2019). Protein-coding variants implicate novel genes related to lipid homeostasis contributing to body-fat distribution. Nat Genet 51(3): 452-469. PubMed ID: 30778226
Body-fat distribution is a risk factor for adverse cardiovascular health consequences. This study analyzed the association of body-fat distribution, assessed by waist-to-hip ratio adjusted for body mass index, with 228,985 predicted coding and splice site variants available on exome arrays in up to 344,369 individuals from five major ancestries (discovery) and 132,177 European-ancestry individuals (validation). Fifteen common (minor allele frequency, MAF >/=5%) and nine low-frequency or rare (MAF <5%) were found coding novel variants. Pathway/gene set enrichment analyses identified lipid particle, adiponectin, abnormal white adipose tissue physiology and bone development and morphology as important contributors to fat distribution, while cross-trait associations highlight cardiometabolic traits. In functional follow-up analyses, specifically in Drosophila RNAi-knockdowns, a significant increase was observed in the total body triglyceride levels for two genes (DNAH10 and PLXND1). This study implicates novel genes in fat distribution, stressing the importance of interrogating low-frequency and protein-coding variants.
Davies, S. A., Cabrero, P., Marley, R., Corrales, G. M., Ghimire, S., Dornan, A. J. and Dow, J. A. T. (2019). Epithelial function in the Drosophila Malpighian tubule: An in vivo renal model. Methods Mol Biol 1926: 203-221. PubMed ID: 30742274
The insect renal (Malpighian) tubule has long been a model system for the study of fluid secretion and its neurohormonal control, as well as studies on ion transport mechanisms. To extend these studies beyond the boundaries of classical physiology, a molecular genetic approach together with the 'omics technologies is required. To achieve this in any vertebrate transporting epithelium remains a daunting task, as the genetic tools available are still relatively unsophisticated. Drosophila melanogaster, however, is an outstanding model organism for molecular genetics. This study describes a technique for fluid secretion assays in the D. melanogaster equivalent of the kidney nephron. The development of this first physiological assay for a Drosophila epithelium, allowing combined approaches of integrative physiology and functional genomics, has now provided biologists with an entirely new model system, the Drosophila Malpighian tubule, which is utilized in multiple fields as diverse as kidney disease research and development of new modes of pest insect control.

Wednesday, March 3rd - Signaling

Guo, P., Lee, C. H., Lei, H., Zheng, Y., Pulgar Prieto, K. D. and Pan, D. (2019). Nerfin-1 represses transcriptional output of Hippo signaling in cell competition. Elife 8. PubMed ID: 30901309
The Hippo tumor suppressor pathway regulates tissue growth in Drosophila by restricting the activity of the transcriptional coactivator Yorkie (Yki), which normally complexes with the TEF/TEAD family DNA-binding transcription factor Scalloped (Sd) to drive the expression of growth-promoting genes. Given its pivotal role as a central hub in mediating the transcriptional output of Hippo signaling, there is great interest in understanding the molecular regulation of the Sd-Yki complex. This study identified Nerfin-1 as a transcriptional repressor that antagonizes the activity of the Sd-Yki complex by binding to the TEA DNA-binding domain of Sd. Consistent with its biochemical function, ectopic expression of Nerfin-1 results in tissue undergrowth in an Sd-dependent manner. Conversely, loss of Nerfin-1 enhances the ability of winner cells to eliminate loser cells in multiple scenarios of cell competition. It was further shown that INSM1, the mammalian ortholog of Nerfin-1, plays a conserved role in repressing the activity of the TEAD-YAP complex. These findings reveal a novel regulatory mode converging on the transcriptional output of the Hippo pathway that may be exploited for modulating the YAP oncoprotein in cancer and regenerative medicine.
Huang, H., Liu, S. and Kornberg, T. B. (2019). Glutamate signaling at cytoneme synapses. Science 363(6430): 948-955. PubMed ID: 30819957
This study investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.
Franco, M. and Carmena, A. (2019). Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells. J Cell Biol. PubMed ID: 30808706
Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. This study uncovered a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. Conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.
Dutta, D., Mutsuddi, M. and Mukherjee, A. (2019). Synergistic interaction of Deltex and Hrp48 leads to JNK activation. Cell Biol Int 43(3): 350-357. PubMed ID: 30597717
The communication among the cells plays a seminal role in metazoan development by coordinating multiple cellular processes that, in turn, helps in the maintenance of biological homeostasis. A previous study demonstrated that deltex (Dx) and Hrp48 together downregulate Notch signaling and induce cell death in Drosophila. To understand the signaling events behind the Dx and Hrp48-induced cell death in a greater detail, a set of genetic experiments was performed followed by immunocytochemical analyses. The data revealed that Dx along with Hrp48 induced JNK activation and consequently cell death in the eye tissue. Additionally, using genetic and molecular approaches, the domain of Dx protein responsible for its synergistic activity with Hrp48 was identified. Altogether, this analyses suggest that coexpression of Dx and Hrp48 activates JNK pathway to induce cell death in eye disc of Drosophila melanogaster.
Johansson, J., Naszai, M., Hodder, M. C., Pickering, K. A., Miller, B. W., Ridgway, R. A., Yu, Y., Peschard, P., Brachmann, S., Campbell, A. D., Cordero, J. B. and Sansom, O. J. (2019). RAL GTPases drive intestinal stem cell function and regeneration through internalization of WNT signalosomes. Cell Stem Cell. PubMed ID: 30853556
Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, this studyidentified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, this study has identified a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
Froldi, F., Pachnis, P., Szuperak, M., Costas, O., Fernando, T., Gould, A. P. and Cheng, L. Y. (2019). Histidine is selectively required for the growth of Myc-dependent dedifferentiation tumours in the Drosophila CNS. Embo j. PubMed ID: 30804004
Rewired metabolism of glutamine in cancer has been well documented, but less is known about other amino acids such as histidine. This study used Drosophila cancer models to show that decreasing the concentration of histidine in the diet strongly inhibits the growth of mutant clones induced by loss of Nerfin-1 or gain of Notch activity. In contrast, changes in dietary histidine have much less effect on the growth of wildtype neural stem cells and Prospero neural tumours. The reliance of tumours on dietary histidine and also on histidine decarboxylase (Hdc) depends upon their growth requirement for Myc. Myc overexpression in nerfin-1 tumours is sufficient to switch their mode of growth from histidine/Hdc sensitive to resistant. This study suggests that perturbations in histidine metabolism selectively target neural tumours that grow via a dedifferentiation process involving large cell size increases driven by Myc.

Tuesday April 2nd - Adult nervous system - Development and function

Ache, J. M., Polsky, J., Alghailani, S., Parekh, R., Breads, P., Peek, M. Y., Bock, D. D., von Reyn, C. R. and Card, G. M. (2019). Neural basis for looming size and velocity encoding in the Drosophila giant fiber escape pathway. Curr Biol 29(6): 1073-1081. PubMed ID: 30827912
Identified neuron classes in vertebrate cortical and subcortical areas and invertebrate peripheral and central brain neuropils encode specific visual features of a panorama. How downstream neurons integrate these features to control vital behaviors, like escape, is unclear. In Drosophila, the timing of a single spike in the giant fiber (GF) descending neuron determines whether a fly uses a short or long takeoff when escaping a looming predator. It has been proposed that GF spike timing results from summation of two visual features whose detection is highly conserved across animals: an object's subtended angular size and its angular velocity. Velocity encoding is attributed to input from lobula columnar type 4 (LC4) visual projection neurons, but the size-encoding source remained unknown. This study shows that lobula plate/lobula columnar, type 2 (LPLC2) visual projection neurons anatomically specialized to detect looming provide the entire GF size component. LPLC2 neurons were found to be necessary for GF-mediated escape, and LPLC2 and LC4 synapse are shown directly onto the GF via reconstruction in a fly brain electron microscopy (EM) volume. LPLC2 silencing eliminates the size component of the GF looming response in patch-clamp recordings, leaving only the velocity component. A model summing a linear function of angular velocity (provided by LC4) and a Gaussian function of angular size (provided by LPLC2) replicates GF looming response dynamics and predicts the peak response time. This study thus presents an identified circuit in which information from looming feature-detecting neurons is combined by a common post-synaptic target to determine behavioral output.
He, L., Gulyanon, S., Mihovilovic Skanata, M., Karagyozov, D., Heckscher, E. S., Krieg, M., Tsechpenakis, G., Gershow, M. and Tracey, W. D. (2019). Direction selectivity in Drosophila proprioceptors requires the mechanosensory channel Tmc. Curr Biol 29(6): 945-956. PubMed ID: 30853433
Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli that produce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. This study used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, it is proposed that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. These findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.
Chahda, J. S., Soni, N., Sun, J. S., Ebrahim, S. A. M., Weiss, B. L. and Carlson, J. R. (2019). The molecular and cellular basis of olfactory response to tsetse fly attractants. PLoS Genet 15(3): e1008005. PubMed ID: 30875383
Dipteran or "true" flies occupy nearly every terrestrial habitat, and have evolved to feed upon a wide variety of sources including fruit, pollen, decomposing animal matter, and even vertebrate blood. This study analyzed the basis of odor response in the tsetse fly Glossina morsitans, which feeds on the blood of humans and their livestock, and is a vector of deadly trypanosomes. The G. morsitans antenna contains specialized subtypes of sensilla, some of which line a sensory pit not found in the fruit fly Drosophila. Distinct patterns of G. morsitans Odor receptor (GmmOr) gene expression in the antenna. A new version of the "empty neuron" heterologous expression system was used it to functionally express several GmmOrs in a mutant olfactory receptor neuron (ORN) of Drosophila. GmmOr35 responds to 1-hexen-3-ol, an odorant found in human emanations, and also alpha-pinene, a compound produced by malarial parasites. Another receptor, GmmOr9, which is expressed in the sensory pit, responds to acetone, 2-butanone and 2-propanol. Acetone and 2-butanone are strong attractants long used in the field to trap tsetse. 2-propanol was found to also be an attractant for both G. morsitans and the related species G. fuscipes, a major vector of African sleeping sickness. The results identify 2-propanol as a candidate for an environmentally friendly and practical tsetse attractant.
Jiang, N., Rasmussen, J. P., Clanton, J. A., Rosenberg, M. F., Luedke, K. P., Cronan, M. R., Parker, E. D., Kim, H. J., Vaughan, J. C., Sagasti, A. and Parrish, J. Z. (2019). A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites. Elife 8. PubMed ID: 30855229
Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. This study describes an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. Somatosensory neurons in Drosophila and zebrafish were found to induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons.
Dag, U., Lei, Z., Le, J. Q., Wong, A., Bushey, D. and Keleman, K. (2019). Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila. Elife 8. PubMed ID: 30801246
Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. This study shows that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. Specific fan-shaped body neurons were identified that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, this study provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation.
Bhukel, A., Beuschel, C. B., Maglione, M., Lehmann, M., Juhasz, G., Madeo, F. and Sigrist, S. J. (2019). Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner. Nat Commun 10(1): 1318. PubMed ID: 30899013
Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. This study shows that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. The results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories.

Monday, April 1st - Stem Cells

Casale, A. M., Cappucci, U., Fanti, L. and Piacentini, L. (2019). Heterochromatin protein 1 (HP1) is intrinsically required for post-transcriptional regulation of Drosophila germline stem cell (GSC) maintenance. Sci Rep 9(1): 4372. PubMed ID: 30867469
A very important open question in stem cells regulation is how the fine balance between germline stem cell (GSC) self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance. Furthermore, recent studies have revealed the importance of HP1 in DNA repair, sister chromatid cohesion and, surprisingly, in positive regulation of gene expression. This study shows that HP1 plays a crucial role in the control of GSC homeostasis in Drosophila. These findings demonstrate that HP1 is required intrinsically to promote GSC self-renewal and progeny differentiation by directly stabilizing the transcripts of key genes involved in GSCs maintenance.
Gultekin, Y. and Steller, H. (2019). Axin proteolysis by Iduna is required for the regulation of stem cell proliferation and intestinal homeostasis in Drosophila. Development 146(6). PubMed ID: 30796047
Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-beta catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna (CG8786) is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/beta-catenin signaling. However, its physiological significance remains to be demonstrated. This study generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.
Verghese, S. and Su, T. T. (2018). Ionizing radiation induces stem cell-like properties in a caspase-dependent manner in Drosophila. PLoS Genet 14(11): e1007659. PubMed ID: 30462636
Cancer treatments including ionizing radiation (IR) can induce cancer stem cell-like properties in non-stem cancer cells, an outcome that can interfere with therapeutic success. Yet, little is understood about the consequences of IR induced stem cell like properties and why some cancer cells show this response but not others. Previous studies have identified a pool of epithelial cells in Drosophila larval wing discs that display IR-induced stem cell-like properties. These cells are resistant to killing by IR and, after radiation damage, change fate and translocate to regenerate parts of the disc that suffered more cell death. This study reports the identification of two new pools of cells with IR-induced regenerative capability. How IR exposure results in the induction of stem cell-like behavior is addressed; a requirement was found for IR-induced caspase activity and for Zfh2, a transcription factor and an effector in the JAK/STAT pathway. Unexpectedly, the requirement for caspase activity was cell-autonomous within cell populations that display regenerative behavior. A model is proposed in which the requirement for caspase activity and Zfh2 can be explained by apoptotic and non-apoptotic functions of caspases in the induction of stem cell-like behavior.
Reedy, A. R., Luo, L., Neish, A. S. and Jones, R. M. (2019). Commensal microbiota induced redox signaling activates proliferative signals in the intestinal stem cell microenvironment. Development. PubMed ID: 30658986
A distinct taxon of the Drosophila microbiota, Lactobacillus plantarum, is capable of stimulating the generation of reactive oxygen species (ROS) within cells, and inducing epithelial cell proliferation. This study shows microbial-induced ROS generation within Drosophila larval stem cell compartments exhibits a distinct spatial distribution. Lactobacilli-induced ROS is strictly excluded from defined midgut compartments that harbor adult midgut progenitor (AMP) cells, forming a functional "ROS sheltered zone" (RSZ). The RSZ is undiscernible in germ-free larvae, but forms following mono-colonization with L. plantarum. L. plantarum is a strong activator of the ROS-sensitive CncC/Nrf2 signaling pathway within enterocytes. Enterocyte-specific activation of CncC stimulated the proliferation of AMPs, demonstrating that pro-proliferative signals are transduced from enterocytes to AMPs. Mechanistically, this study shows that the cytokine Upd2 is expressed in the gut following L. plantarum colonization in a CncC dependent fashion, and may function in lactobacilli-induced AMP proliferation and intestinal tissue growth and development.
Otsuki, L. and Brand, A. H. (2019). Dorsal-ventral differences in neural stem cell quiescence are induced by p57(KIP2)/Dacapo. Dev Cell. PubMed ID: 30905769
Quiescent neural stem cells (NSCs) in the adult brain are regenerative cells that could be activated therapeutically to repair damage. It is becoming apparent that quiescent NSCs exhibit heterogeneity in their propensity for activation and in the progeny that they generate. NSCs have been shown to undergo quiescence in either G0 or G2 in the Drosophila brain, challenging the notion that all quiescent stem cells are G0 arrested. G2-quiescent NSCs become activated prior to G0 NSCs. This study shows that the cyclin-dependent kinase inhibitor Dacapo (Dap; ortholog of p57(KIP2)) determines whether NSCs enter G0 or G2 quiescence during embryogenesis. The dorsal patterning factor Muscle segment homeobox (Msh; ortholog of MSX1/2/3) binds directly to the Dap locus and induces Dap expression in dorsal NSCs, resulting in G0 arrest, while more ventral NSCs undergo G2 quiescence. These results reveal region-specific regulation of stem cell quiescence.
Wu, D., Wu, L., An, H., Bao, H., Guo, P., Zhang, B., Zheng, H., Zhang, F., Ge, W., Cai, Y., Xi, Y. and Yang, X. (2018). RanGAP-mediated nucleocytoplasmic transport of Prospero regulates neural stem cell lifespan in Drosophila larval central brain. Aging Cell: e12854. PubMed ID: 30549175
By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. This study reveals that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran-mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one-way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. These data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. This study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.
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