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Current papers in developmental biology and gene function


Friday, February 30th, 2020 - Stem cells

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Xu, C., Franklin, B., Tang, H. W., Regimbald-Dumas, Y., Hu, Y., Ramos, J., Bosch, J. A., Villalta, C., He, X. and Perrimon, N. (2019). An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity. Proc Natl Acad Sci U S A. PubMed ID: 31852821
Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, this study identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, this study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation.
Tiwari, M. D., Zeitler, D. M., Meister, G. and Wodarz, A. (2019). Molecular profiling of stem cell-like female germ line cells in Drosophila delineates networks important for stemness and differentiation. Biol Open 8(11). PubMed ID: 31649115
Stem cells can self-renew and produce daughter cells destined for differentiation. The precise control of the balance between these two outcomes is essential to ensure tissue homeostasis and to prevent uncontrolled proliferation resulting in tumor formation. As self-renewal and differentiation are likely to be controlled by different gene expression programs, unraveling the underlying gene regulatory networks is crucial for understanding the molecular logic of this system. This study has characterized by next generation RNA sequencing (RNA-seq) the transcriptome of germline stem cell (GSC)-like cells isolated from bag of marbles (bam) mutant Drosophila ovaries and compared it to the transcriptome of germ line cells isolated from wild-type ovaries. This dataset was complemented by utilizing an RNA-immunoprecipitation strategy to identify transcripts bound to the master differentiation factor Bam. Protein complex enrichment analysis on these combined datasets allows delineation of known and novel networks essential for GSC maintenance and differentiation. Further comparative transcriptomics illustrates similarities between GSCs and primordial germ cells and provides a molecular footprint of the stem cell state. This study represents a useful resource for functional studies on stem cell maintenance and differentiation.
Zhao, S., Wu, C., Gao, Z., Li, X., Zheng, G. and Wang, Z. (2019). Notch signaling governs the expression of glypican Dally to define the stem cell niche. Biol Open. PubMed ID: 31826854
Extracellular glypicans play pivotal roles in organogenesis, stem cell maintenance, and cancer development. However, the growth phenotypes associated with different levels of glypican are not consistent in development or tumorigenesis. This requires the clarification on how the spatial patterns of glypican relate to the distribution of signaling molecules in different cellular context, and how glypican expression is regulated. Previous work has shown that Dally, one of the glypican members in Drosophila, is required in the niche for the maintenance of germline stem cells (GSCs) via short-range BMP signaling in ovary. However, the regulatory mechanism of glypican pattern in the ovarian stem cell niche remains elusive. The current data demonstrate that Notch pathway is genetically upstream of Dally and its function to maintain GSC relies on Dally expression. Combining yeast and fruit fly genetics, this study illustrates that Dally is under the transcriptional control of Notch signaling via the transcription factor Su(H). Further, human glypicans and disease-associated variants were assayed in Drosophila ovary, which can serve as an effective system to evaluate the structure-function relationship of the human homologs.
Kwon, Y. V., Zhao, B., Xu, C., Lee, J., Chen, C. L., Vinayagam, A., Edgar, B. A. and Perrimon, N. (2019). The role of translationally controlled tumor protein in proliferation of Drosophila intestinal stem cells. Proc Natl Acad Sci U S A. PubMed ID: 31843907
Translationally controlled tumor protein (TCTP) is a highly conserved protein functioning in multiple cellular processes, ranging from growth to immune responses. To explore the role of TCTP in tissue maintenance and regeneration, this study employed the adult Drosophila midgut, where multiple signaling pathways interact to precisely regulate stem cell division for tissue homeostasis. Tctp levels were significantly increased in stem cells and enteroblasts upon tissue damage or activation of the Hippo pathway that promotes regeneration of intestinal epithelium. Stem cells with reduced Tctp levels failed to proliferate during normal tissue homeostasis and regeneration. Mechanistically, Tctp forms a complex with multiple proteins involved in translation and genetically interacts with ribosomal subunits. In addition, Tctp increases both Akt1 protein abundance and phosphorylation in vivo. Altogether, Tctp regulates stem cell proliferation by interacting with key growth regulatory signaling pathways and the translation process in vivo.
Endow, S. A., Miller, S. E. and Ly, P. T. (2019). Mitochondria-enriched protrusions are associated with brain and intestinal stem cells in Drosophila. Commun Biol 2: 427. PubMed ID: 31799429
Brain stem cells stop dividing in late Drosophila embryos and begin dividing again in early larvae after feeding induces reactivation. Quiescent neural stem cells (qNSCs) display an unusual cytoplasmic protrusion that is no longer present in reactivated NSCs. The protrusions join the qNSCs to the neuropil, brain regions that are thought to maintain NSCs in an undifferentiated state, but the function of the protrusions is not known. This study shows that qNSC protrusions contain clustered mitochondria that are likely maintained in position by slow forward-and-backward microtubule growth. Larvae treated with a microtubule-stabilizing drug show bundled microtubules and enhanced mitochondrial clustering in NSCs, together with reduced qNSC reactivation. It was further shown that intestinal stem cells contain mitochondria-enriched protrusions. The qNSC and intestinal stem-cell protrusions differ from previously reported cytoplasmic extensions by forming stem-cell-to-niche mitochondrial bridges that could potentially both silence genes and sense signals from the stem cell niche.
Hao, X., Wang, S., Lu, Y., Yu, W., Li, P., Jiang, D., Guo, T., Li, M., Li, J., Xu, J., Wu, W., Ho, M. S. and Zhang, L. (2020). Lola regulates Drosophila adult midgut homeostasis via non-canonical hippo signaling. Elife 9. PubMed ID: 31934851
Tissue homeostasis and regeneration in the Drosophila midgut is regulated by a diverse array of signaling pathways including the Hippo pathway. Hippo signaling restricts intestinal stem cell (ISC) proliferation by sequestering the transcription co-factor Yorkie (Yki) in the cytoplasm, a factor required for rapid ISC proliferation under injury-induced regeneration. Nonetheless, the mechanism of Hippo-mediated midgut homeostasis and whether canonical Hippo signaling is involved in ISC basal proliferation are less characterized. This study identified Lola as a transcription factor acting downstream of Hippo signaling to restrict ISC proliferation in a Yki-independent manner. Not only that, Lola interacts with and is stabilized by the Hippo signaling core kinase Warts (Wts), Lola rescues the enhanced ISC proliferation upon Wts depletion via suppressing Dref and SkpA expressions. These findings reveal that Lola is a non-canonical Hippo signaling component in regulating midgut homeostasis, providing insights on the mechanism of tissue maintenance and intestinal function.

Thursday, February 27th - Chromatin

Ghotbi, E., Lackey, K., Wong, V., Thompson, K. T., Caston, E. G., Haddadi, M., Benes, J. and Jones, R. S. (2020). Differential contributions of DNA binding proteins to Polycomb response element activity at the Drosophila giant gene. Genetics. PubMed ID: 31919108
Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic regulators whose primary function is to maintain the transcriptional repression of target genes. Recruitment of Drosophila melanogaster PcG proteins to target genes requires the presence of one or more Polycomb Response Elements (PREs). The functions or necessity for more than one PRE at a gene are not clear and individual PREs at some loci may have distinct regulatory roles. Various combinations of sequence-specific DNA binding proteins are present at a given PRE, but only Pleiohomeotic (Pho) is present at all strong PREs. The giant (gt) locus has two PREs, a proximal PRE1 and a distal PRE2. During early embryonic development, Pho binds to PRE1 approximately 90 minutes prior to stable binding to PRE2. This observation indicated a possible dependence of PRE2 on PRE1 for PcG recruitment; however, this study finds that PRE2 recruits PcG proteins and maintains transcriptional repression independently of Pho binding to PRE1. Pho-like (Phol) is partially redundant with Pho during larval development and binds to the same DNA sequences in vitro. Although binding of Pho to PRE1 is dependent on the presence of consensus Pho-Phol binding sites, Phol binding is less so and appears to play a minimal role in recruiting other PcG proteins to gt. Another PRE binding protein, Spps, is dependent on the presence of Pho for PRE1 binding. Further, this study showed that, in addition to silencing gene expression, PcG proteins dampen transcription of an active gene.
Ekhteraei-Tousi, S., Lewerentz, J. and Larsson, J. (2020). Painting of fourth and the X-linked 1.688 satellite in D. melanogaster is involved in chromosome-wide gene regulation. Cells 9(2). PubMed ID: 32019091
Chromosome-specific regulatory mechanisms provide a model to understand the coordinated regulation of genes on entire chromosomes or on larger genomic regions. In fruit flies, two chromosome-wide systems have been characterized: The male-specific lethal (MSL) complex, which mediates dosage compensation and primarily acts on the male X-chromosome, and Painting of fourth (POF), which governs chromosome-specific regulation of genes located on the 4th chromosome. How targeting of one specific chromosome evolves is still not understood; but repeated sequences, in forms of satellites and transposable elements, are thought to facilitate the evolution of chromosome-specific targeting. The highly repetitive 1.688 satellite has been functionally connected to both these systems. Considering the rapid evolution and the necessarily constant adaptation of regulatory mechanisms, such as dosage compensation, it is hypothesised that POF and/or 1.688 may still show traces of dosage-compensation functions. This hypothesis was tested by transcriptome analysis. Loss of Pof decreases not only chromosome 4 expression but also reduces the X-chromosome expression in males. The 1.688 repeat deletion, Zhr(1) (Zygotic hybrid rescue), does not affect male dosage compensation detectably; however, Zhr(1) in females causes a stimulatory effect on X-linked genes with a strong binding affinity to the MSL complex (genes close to high-affinity sites). Lack of pericentromeric 1.688 also affected 1.688 expression in trans and was linked to the differential expression of genes involved in eggshell formation. These results are discussed with reference to the connections between POF, the 1.688 satellite and dosage compensation, and the role of the 1.688 satellite in hybrid lethality.
Arzate-Mejia, R. G., Josue Cerecedo-Castillo, A., Guerrero, G., Furlan-Magaril, M. and Recillas-Targa, F. (2020). In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila. Nat Commun 11(1): 894. PubMed ID: 32060283
Chromosomes are organized into high-frequency chromatin interaction domains called topologically associating domains (TADs), which are separated from each other by domain boundaries. The molecular mechanisms responsible for TAD formation are not yet fully understood. In Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by architectural proteins (APs) remains controversial. This study investigated the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch gene locus in Drosophila. Deletion of domain boundaries was found to result in TAD fusion and long-range topological defects that are accompanied by loss of APs and RNA Pol II chromatin binding as well as defects in transcription. Together, these results provide compelling evidence of the contribution of discrete genetic sequences bound by APs and RNA Pol II in the partition of the genome into TADs and in the regulation of gene expression in Drosophila.
Bayer, F. E., Deichsel, S., Mahl, P. and Nagel, A. C. (2020). Drosophila Xrcc2 regulates DNA double-strand repair in somatic cells. DNA Repair (Amst) 88: 102807. PubMed ID: 32006716
Genomic integrity is challenged by endo- and exogenous assaults that are combated by highly conserved DNA repair mechanisms. High fidelity recombination repair of DSBs relies on the Rad51 recombinase, aided by several Rad51 paralogs. Only two out of four Rad51 paralogs in Drosophila have been studied so far and both are restricted to meiotic recombination repair. Using CRISPR/Cas9 technology, this study has generated the first X-ray repair cross complementing 2 (xrcc2) null mutant in Drosophila. Like any other Drosophila Rad51 homologue, loss of xrcc2 does not affect fly development. Drosophila xrcc2 - despite a specific expression in ovaries - is not essential for meiotic DSB repair, but supports the process. In contrast, xrcc2 is required for mitotic DNA damage repair: the mutants are highly sensitive towards various genotoxic stressors, including ionizing radiation, which significantly increase mortality. Moreover, loss of xrcc2 provokes chromosome aberrations in mitotic larval neuroblasts under unstressed conditions and enduring chromosomal breaks as well as persistent repair foci after irradiation exposure. Together these results demonstrate that xrcc2 plays a crucial role in combating genotoxic insult by controlling DSB repair in somatic cells of Drosophila.
Bonchuk, A., Kamalyan, S., Mariasina, S., Boyko, K., Popov, V., Maksimenko, O. and Georgiev, P. (2020). N-terminal domain of the architectural protein CTCF has similar structural organization and ability to self-association in bilaterian organisms. Sci Rep 10(1): 2677. PubMed ID: 32060375
CTCF is the main architectural protein found in most of the examined bilaterian organisms. The cluster of the C2H2 zinc-finger domains involved in recognition of long DNA-binding motif is only part of the protein that is evolutionarily conserved, while the N-terminal domain (NTD) has different sequences. Biophysical characterization was carried out of CTCF NTDs from various species representing all major phylogenetic clades of higher metazoans. With the exception of Drosophilides, the N-terminal domains of CTCFs show an unstructured organization and absence of folded regions in vitro. In contrast, NTDs of Drosophila melanogaster and virilis CTCFs contain unstructured folded regions that form tetramers and dimers correspondingly in vitro. Unexpectedly, most NTDs are able to self-associate in the yeast two-hybrid and co-immunoprecipitation assays. These results suggest that NTDs of CTCFs might contribute to the organization of CTCF-mediated long-distance interactions and chromosomal architecture.
Di Mauro, G., Carbonell, A., Escudero-Ferruz, P. and Azorin, F. (2020). The zinc-finger proteins WOC and ROW play distinct functions within the HP1c transcription complex. Biochim Biophys Acta Gene Regul Mech: 194492. PubMed ID: 32006714
In Drosophila, the Heterochromatin Protein 1c (HP1c) forms a transcriptional complex with the zinc-finger proteins WOC and ROW, and the extraproteasomal ubiquitin receptor Dsk2. This complex localizes at promoters of active genes and it is required for transcription. The functions played by the different components of the HP1c complex are not fully understood. This study shows that WOC and ROW are required for chromatin binding of both Dsk2 and HP1c. However, while impairing chromatin binding strongly destabilizes HP1c, it does not affect Dsk2 stability. It was also shown that WOC, but not ROW, is required for nuclear localization of Dsk2. Moreover, WOC and Dsk2 co-immunoprecitate upon ROW depletion. These results suggest that WOC and Dsk2 interact to form a subcomplex that mediates nuclear translocation of Dsk2. This study also shows that ROW mediates chromatin binding of the WOC/Dsk2 subcomplex, as well as of HP1c. Altogether these observations favor a model by which the interaction with WOC recruits Dsk2 to the HP1c complex that, in its turn, binds chromatin in a ROW-dependent manner.

Wednesday, February 26th - Gonad development

Luo, J., Zhou, P., Guo, X., Wang, D. and Chen, J. (2019). The polarity protein Dlg5 regulates collective cell migration during Drosophila oogenesis. PLoS One 14(12): e0226061. PubMed ID: 31856229
Collective migration plays critical roles in animal development, physiological events, and cancer metastasis. However, the molecular mechanisms of collective cell migration are not well understood. Drosophila border cells represent an excellent in vivo genetic model to study collective cell migration and identify novel regulatory genes for cell migration. Using the Mosaic Analysis with a Repressible Cell Marker (MARCM) system, 240 P-element insertion lines were screened to identify essential genes for border cell migration. Two genes were uncovered, including dlg5 (discs large 5) and CG31689. Further analysis showed that Dlg5 regulates the apical-basal polarity and cluster integrity in border cell clusters. Dlg5 is enriched in lateral surfaces between border cells and central polar cells but also shows punctate localization between border cells. The distribution of Dlg5 in border cell clusters is regulated by Armadillo. Structure-function analysis revealed that the N-terminal Coiled-coil domain and the C-terminal PDZ3-PDZ4-SH3-GUK domains but not the PDZ1-PDZ2 domains of Dlg5 are required for BC migration. The Coiled-coil domain and the PDZ4-SH3-GUK domains are critical for Dlg5's cell surface localization in border cell clusters.
Hopkins, et al. (2019). BMP signaling inhibition in Drosophila secondary cells remodels the seminal proteome and self and rival ejaculate functions. Proc Natl Acad Sci U S A 116(49): 24719-24728. PubMed ID: 31740617
Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. In Drosophila, most SFPs are produced in the accessory glands, which are composed of ~1,000 fertility-enhancing "main cells" and ~40 more functionally cryptic "secondary cells." Inhibition of BMP signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might regulate global SFP functions and proteome composition. Secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Changes to the seminal proteome were revealed that encompass alterations to main-cell-derived proteins, indicating important cross-talk between classes of SFP-secreting cells. These results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution.
York-Andersen, A. H., Hu, Q., Wood, B. W., Wolfner, M. F. and Weil, T. T. (2019). A calcium-mediated actin redistribution at egg activation in Drosophila. Mol Reprod Dev. PubMed ID: 31880382
Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. This study used imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. Before egg activation, actin was found to be smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, actin spreading out as the egg swells though the intake of fluid. A relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, these data show that calcium resets the actin cytoskeleton at egg activation, a model that is proposed to be likely conserved in other species.
Fang, J. and Lerit, D. A. (2019). Drosophila pericentrin-like protein promotes the formation of primordial germ cells. Genesis: e23347. PubMed ID: 31774613
Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. Previous work has identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin-like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. This study identified a role for PLP at several discrete steps during PGC development. PLP was found to function in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.
Lovegrove, H. E., Bergstralh, D. T. and St Johnston, D. (2019). The role of integrins in Drosophila egg chamber morphogenesis. Development 146(23). PubMed ID: 31784458
The Drosophila egg chamber comprises a germline cyst surrounded by a tightly organised epithelial monolayer, the follicular epithelium (FE). Loss of integrin function from the FE disrupts epithelial organisation at egg chamber termini, but the cause of this phenotype remains unclear. This study shows that the beta-integrin Myospheroid (Mys) is only required during early oogenesis when the pre-follicle cells form the FE. Mutation of mys disrupts both the formation of a monolayered epithelium at egg chamber termini and the morphogenesis of the stalk between adjacent egg chambers, which develops through the intercalation of two rows of cells into a single-cell-wide stalk. Secondary epithelia, like the FE, have been proposed to require adhesion to the basement membrane to polarise. However, Mys is not required for pre-follicle cell polarisation, as both follicle and stalk cells localise polarity factors correctly, despite being mispositioned. Instead, loss of integrins causes pre-follicle cells to constrict basally, detach from the basement membrane and become internalised. Thus, integrin function is dispensable for pre-follicle cell polarity but is required to maintain cellular organisation and cell shape during morphogenesis.
Di Giorgio, M. L., Morciano, P., Bucciarelli, E., Porrazzo, A., Cipressa, F., Saraniero, S., Manzi, D., Rong, Y. S. and Cenci, G. (2020). The Drosophila citrate lyase is required for cell division during spermatogenesis. Cells 9(1). PubMed ID: 31947614
The Drosophila melanogaster ATPCL gene encodes for the human ATP Citrate Lyase (ACL) ortholog, a metabolic enzyme that from citrate generates glucose-derived Acetyl-CoA, which fuels central biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine, and the acetylation of proteins and histones. Although loss of Drosophila ATPCL reduces levels of Acetyl-CoA, unlike its human counterpart, it does not affect global histone acetylation and gene expression, suggesting that its role in histone acetylation is either partially redundant in Drosophila or compensated by alternative pathways. This study describes that depletion of ATPCL affects spindle organization, cytokinesis, and fusome assembly during male meiosis, revealing an unanticipated role for ATPCL during spermatogenesis. ATPCL mutant meiotic phenotype is in part caused by a reduction of fatty acids, but not of triglycerides or cholesterol, indicating that ATPCL-derived Acetyl-CoA is predominantly devoted to the biosynthesis of fatty acids during spermatogenesis. Collectively, these results unveil for the first time an involvement for ATPCL in the regulation of meiotic cell division, which is likely conserved in human cells.

Tuesday, February 25th - Disease Models

Ishikawa, K. and Nagai, Y. (2019). Molecular Mechanisms and Future Therapeutics for Spinocerebellar Ataxia Type 31 (SCA31). Neurotherapeutics. PubMed ID: 31755042
Spinocerebellar ataxia type 31 (SCA31) is one of the autosomal-dominant neurodegenerative disorders that shows progressive cerebellar ataxia as a cardinal symptom. This disease is caused by a 2.5- to 3.8-kb-long complex pentanucleotide repeat containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n in an intron of the gene called BEAN1 (brain expressed, associated with Nedd4). By comparing various pentanucleotide repeats in this particular locus among control Japanese and Caucasian populations, it was found that (TGGAA)n was the only sequence segregating with SCA31, strongly suggesting the pathogenicity of (TGGAA)n. The complex repeat also lies in an intron of another gene, TK2 (thymidine kinase 2), which is transcribed in the opposite direction, indicating that the complex repeat is bi-directionally transcribed as noncoding repeats. In SCA31 human brains, (UGGAA)n, the BEAN1 transcript of SCA31 mutation was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. Subsequent RNA pulldown analysis disclosed that (UGGAA)n binds to RNA-binding proteins TDP-43, FUS, and hnRNP A2/B1. In fact, TDP-43 was found to co-localize with RNA foci in human SCA31 Purkinje cells. To dissect the pathogenesis of (UGGAA)n in SCA31, transgenic fly models of SCA31 were generated by overexpressing SCA31 complex pentanucleotide repeats in Drosophila. The toxicity of (UGGAA)n was found to be length- and expression level-dependent, and it was dampened by co-expressing TDP-43, FUS, and hnRNP A2/B1. Further investigation revealed that TDP-43 ameliorates (UGGAA)n toxicity by directly fixing the abnormal structure of (UGGAA)n. This led to a proposal that TDP-43 acts as an RNA chaperone against toxic (UGGAA)n. Further research on the role of RNA-binding proteins as RNA chaperones may provide a novel therapeutic strategy for SCA31.
Kampf, L. L. et al. (2019). TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome. J Am Soc Nephrol 30(12): 2338-2353. PubMed ID: 31732614
Mutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology. This study used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). The functional significance was analyzed of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes. Hemizygous missense mutations were identified in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function. It is concluded that novel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. These findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.
Napoli, B., Gumeni, S., Forgiarini, A., Fantin, M., De Filippis, C., Panzeri, E., Vantaggiato, C. and Orso, G. (2019). Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes. Front Neurosci 13: 1202. PubMed ID: 31803000
Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. This study investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. The data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. These data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment.
Chongtham, A. et al. (2020). Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT. Hum Mol Genet. PubMed ID: 31943010
Huntington's Disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the Huntingtin protein (HTT) and is necessary to cause pathology and formation of HTT aggregates. This study asked whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, the effects were compared of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.
Delrio-Lorenzo, A., Rojo-Ruiz, J., Alonso, M. T. and Garcia-Sancho, J. (2020). Sarcoplasmic reticulum Ca(2+) decreases with age and correlates with the decline in muscle function in Drosophila. J Cell Sci. PubMed ID: 32005702
Sarcopenia, the loss of muscle mass and strength associated to age, has been linked to impairment of the cytosolic Ca(2+) peak that triggers muscle contraction, but mechanistic details remain unknown. This study explored the hypothesis that a reduction in sarcoplasmic reticulum Ca(2+) concentration ([Ca(2+)]SR) is at the origin of this loss of Ca(2+) homeostasis. Drosophila melanogaster was engineered to express the Ca(2+) indicator GAP3 targeted to muscle SR, and a new method was developed to calibrate the signal into [Ca(2+)]SR. In vivo [Ca(2+)]SR dropped with age from approximately 600 microM down to 50 microM in close correlation to muscle function, which declined monotonically when [Ca(2+)]SR was <400 microM. [Ca(2+)]SR results from the pump-leak steady-state at the SR membrane. However, changes in expression of the SERCA pump and of the ryanodine receptor leak, were too modest to explain the large changes seen in [Ca(2+)]SR. Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca(2+)]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca(2+)] with age in brain neurons.
Bergkvist, L., Richards, D. R., Bernardo-Gancedo, A., Kumita, J. R., Nilsson, P. R. and Brorsson, A. C. (2020). Serum amyloid P component promotes formation of distinct aggregated lysozyme morphologies and reduces toxicity in Drosophila flies expressing F57I lysozyme. PLoS One 15(1): e0227227. PubMed ID: 31978114
Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using a Drosophila model of systemic lysozyme amyloidosis, SAP has been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. It was found that SAP can counteract the toxicity induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.

Monday February 24th - Adult neural development and function

Clarke, A., McQueen, P. G., Fang, H. Y., Kannan, R., Wang, V., McCreedy, E., Buckley, T., Johannessen, E. M., Wincovitch, S. and Giniger, E. (2020). Dynamic morphogenesis of a pioneer axon in Drosophila and its regulation by Abl tyrosine kinase. Mol Biol Cell: mbcE19100563. PubMed ID: 31967935
The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. This study dissected this process using live imaging of the TSM1 axon of the developing Drosophila wing. The growth cone is almost purely filopodial, and it extends by a protrusive mode of growth. Quantitative analysis reveals two separate groups of growth cone properties that together account for growth cone structure and dynamics. The core morphological features of the growth cone are strongly correlated with one another and define two discrete morphs. Genetic manipulation of a critical mediator of axon guidance signaling, Abl tyrosine kinase, shows that while Abl weakly modulates the ratio of the two morphs it does not greatly change their properties. Rather, Abl primarily regulates the second group of properties, which report the organization and distribution of actin in the growth cone, and are coupled to growth cone velocity. In an accompanying paper dissects the nature of that regulation of actin organization and how it controls the spatial localization of filopodial dynamics, and thus axon extension. Together, these observations suggest a novel, probabilistic mechanism by which Abl biases the stochastic fluctuations of growth cone actin to direct axon growth and guidance.
Clarke, A., McQueen, P. G., Fang, H. Y., Kannan, R., Wang, V., McCreedy, E., Wincovitch, S. and Giniger, E. (2020). Abl signaling directs growth of a pioneer axon in Drosophila by shaping the intrinsic fluctuations of actin. Mol Biol Cell: mbcE19100564. PubMed ID: 31967946
The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. In an accompanying paper, live imaging of the TSM1 axon of the developing Drosophila wing was use to show that the essential role of the core guidance signaling molecule, Abl tyrosine kinase, is to modulate the organization and spatial localization of actin in the advancing growth cone. This study dissects in detail the properties of that actin organization, and its consequences for growth cone morphogenesis and motility. Sdvance of the actin mass in the distal axon is shown to drive the forward motion of the dynamic filopodial domain that defines the growth cone. It was further shown that Abl regulates both the width of the actin mass and its internal organization, spatially biasing the intrinsic fluctuations of actin to achieve net advance of the actin, and thus of the dynamic filopodial domain of the growth cone, while maintaining the essential coherence of the actin mass itself. These data suggest a model whereby guidance signaling systematically shapes the intrinsic, stochastic fluctuations of actin in the growth cone to produce axon growth and guidance.
Rozenfeld, E., Lerner, H. and Parnas, M. (2019). Muscarinic modulation of antennal lobe GABAergic local neurons shapes odor coding and behavior. Cell Rep 29(10): 3253-3265.e3254. PubMed ID: 31801087
In the antennal lobe (AL), the first olfactory relay of Drosophila, excitatory neurons are predominantly cholinergic. Ionotropic nicotinic receptors play a vital role in the effects of acetylcholine in the AL. However, the AL also has a high expression level of metabotropic muscarinic acetylcholine receptors type A (mAChRs-A). Nevertheless, the neurons expressing them and their role in the AL are unknown. Elucidating their function may reveal principles in olfactory modulation. This study shows that mAChRs-A shape AL output and affect behavior. Effects of mAChRs-A were localized to a sub-population of GABAergic local neurons (iLNs), where they play a dual role: direct excitation of iLNs and stabilization of the synapse between receptor neurons and iLNs, which undergoes strong short-term depression. These results reveal modulatory functions of the AL main excitatory neurotransmitter. Striking similarities to the mammalian olfactory system predict that mammalian glutamatergic metabotropic receptors could be associated with similar modulations.
Chia, J. and Scott, K. (2020). Activation of specific mushroom body output neurons inhibits proboscis extension and sucrose consumption. PLoS One 15(1): e0223034. PubMed ID: 31990947
In Drosophila, the mushroom bodies (MBs) are critical for olfactory associative learning and conditioned taste aversion, but how the output of the MBs affects specific behavioral responses is unresolved. In conditioned taste aversion, Drosophila shows a specific behavioral change upon learning: proboscis extension to sugar is reduced after a sugar stimulus is paired with an aversive stimulus. While studies have identified MB output neurons (MBONs) that drive approach or avoidance behavior, whether the same MBONs impact innate proboscis extension behavior is unknown. This study tested the role of MB pathways in altering proboscis extension and identified MBONs that synapse onto multiple MB compartments that upon activation significantly decreased proboscis extension to sugar. Activating several of these lines also decreased sugar consumption, revealing that these MBONs have a general role in modifying feeding behavior beyond proboscis extension. The MBONs that decreased proboscis extension and ingestion are different from those that drive avoidance behavior in another context. These studies provide insight into how activation of MB output neurons decreases proboscis extension to taste compounds.
Scheunemann, L., Lampin-Saint-Amaux, A., Schor, J. and Preat, T. (2019). A sperm peptide enhances long-term memory in female Drosophila. Sci Adv 5(11): eaax3432. PubMed ID: 31799390
Can mating influence cognitive functions such as learning and memory in a permanent way? This question was addressed using a combined behavioral and in vivo imaging approach, finding that aversive long-term memory performance strongly increases in Drosophila females in response to sperm transfer following mating. A peptide in the male sperm, the sex peptide, is known to cause marked changes in female reproductive behavior, as well as other behaviors such as dietary preference. This study demonstrates that sex peptide enhances memory by acting on a single pair of serotonergic brain neurons, in which activation of the sex peptide receptor stimulates the cyclic adenosine monophosphate/protein kinase A pathway. This study thus reveals a strong effect of mating on memory via the neuromodulatory action of a sperm peptide on the female brain.
Sen, R., Wang, K. and Dickson, B. J. (2019). TwoLumps ascending neurons mediate touch-evoked reversal of walking direction in Drosophila. Curr Biol 29(24): 4337-4344. PubMed ID: 31813606
External cues, including touch, enable walking animals to flexibly maneuver around obstacles and extricate themselves from dead-ends. In a screen for neurons that enable Drosophila melanogaster to retreat when it encounters a dead-end, this study identified a pair of ascending neurons, the TwoLumps Ascending (TLA) neurons. Silencing TLA activity impairs backward locomotion, whereas optogenetic activation triggers backward walking. TLA-induced reversal is mediated in part by the Moonwalker Descending Neurons (MDNs), which receive excitatory input from the TLAs. Silencing the TLAs decreases the extent to which freely walking flies back up upon encountering a physical barrier in the dark, and TLAs show calcium responses to optogenetic activation of neurons expressing the mechanosensory channel NOMPC. It is infered that TLAs convey feedforward mechanosensory stimuli to transiently activate MDNs in response to anterior body touch.

Friday, February 21st - Adult Development

Jiang, Y. F., Lin, H. L., Wang, L. J., Hsu, T. and Fu, C. Y. (2020). Coordinated organization of mitochondrial lamellar cristae and gain of COX function during mitochondrial maturation in Drosophila. Mol Biol Cell 31(1): 18-26. PubMed ID: 31746672
Mitochondrial cristae contain electron transport chain complexes and are distinct from the inner boundary membrane (IBM). While many details regarding the regulation of mitochondrial structure are known, the relationship between cristae structure and function during organelle development is not fully described. This study used serial-section tomography to characterize the formation of lamellar cristae in immature mitochondria during a period of dramatic mitochondrial development that occurs after Drosophila emergence as an adult. The formation of lamellar cristae was associated with the gain of cytochrome c oxidase (COX) function, and the COX subunit, COX4, was localized predominantly to organized lamellar cristae. Interestingly, 3D tomography showed some COX-positive lamellar cristae were not connected to IBM. It is hypothesized that some lamellar cristae may be organized by a vesicle germination process in the matrix, in addition to invagination of IBM. OXA1 protein, which mediates membrane insertion of COX proteins, was also localized to cristae and reticular structures isolated in the matrix additional to the IBM, suggesting that it may participate in the formation of vesicle germination-derived cristae. Overall, this study elaborates on how cristae morphogenesis and functional maturation are intricately associated. These data support the vesicle germination and membrane invagination models of cristae formation.
Choubey, P. K., Nandy, N., Pandey, A. and Roy, J. K. (2020). Rab11 plays a key role in stellate cell differentiation via non-canonical Notch pathway in Malpighian tubules of Drosophila melanogaster. Dev Biol. PubMed ID: 31911183
This study reports a novel role of Rab11 in the differentiation of stellate cells via the non-canonical Notch pathway in Malpighian tubules. During Malpighian tubule development caudal visceral mesodermal cells intercalate into the epithelial tubule of ectodermal origin consisting of principal cells, undergo mesenchymal to epithelial transition and differentiate into star shaped stellate cells in adult Malpighian tubule. Two transcription factors, Teashirt and Cut (antagonistic to each other) are known to be expressed in stellate cells and principal cells, respectively, from early stages of development and serve as markers for these cells. Inhibition of Rab11 function or over-expression of activated Notch in stellate cells resulted in the expression of Cut that leads to down-regulation of Teashirt or vice-versa that leads to hampered differentiation of stellate cells. The stellate cells do not transform to star/bar shaped and remain in mesenchymal state in adult Malpighian tubule. Over-expression of Deltex, which plays important role in non-canonical Notch signaling pathway, shows similar phenotype of stellate cells as seen in individuals with down-regulated Rab11, while down-regulation of Deltex in genetic background of Rab11(RNAi) rescues Teashirt expression and shape of stellate cells. These experiments suggest that an inhibition or reduction of Rab11 function in stellate cells results in the faulty recycling of Notch receptors to plasma membrane as they accumulate in early and late endosomes, leading to Deltex mediated non-canonical Notch activation.
Simon, E., de la Puebla, S. F. and Guerrero, I. (2019). Drosophila Zic family member odd-paired is needed for adult post-ecdysis maturation. Open Biol 9(12): 190245. PubMed ID: 31847787
Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In Drosophila melanogaster, the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). The role of the transcription factor Odd-paired (Opa) during the post-eclosion period. opa is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innervating the larval muscles involved in adult ecdysis.Its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologue Zic2, in the CCAP neurons also affects Burs expression, indicating an evolutionary functional conservation. Finally, the results show that, independently of its role in Burs regulation, Opa prevents death of CCAP neurons during larval development.
Best, B. T. and Leptin, M. (2020). Multiple Requirements for Rab GTPases in the Development of Drosophila Tracheal Dorsal Branches and Terminal Cells. G3 (Bethesda). PubMed ID: 31980432
Tracheal cells use Rab GTPases to organize their internal membrane transport, resulting in the specific localization or secretion of proteins on the apical or basal membrane compartments. Some contributions of Rabs to junctional remodelling and governance of tracheal lumen contents are known, but it is reasonable to assume that they play important further roles in morphogenesis. This pertains in particular to terminal tracheal cells, specialized branch-forming cells that drastically reshape both their apical and basal membrane during the larval stages. A loss-of-function screen was performed in the tracheal system, knocking down endogenously tagged alleles of 26 Rabs by targeting the tag via RNAi. This revealed that at least 14 Rabs are required to ensure proper cell fate specification and migration of the dorsal branches, as well as their epithelial fusion with the contralateral dorsal branch. The screen implicated four Rabs in the subcellular morphogenesis of terminal cells themselves. Further tests suggested residual gene function after knockdown, leading to a discussion the limitations of this approach. It is concluded that more Rabs than identified here may be important for tracheal morphogenesis, and that the tracheal system offers great opportunities for studying several Rabs that have barely been characterized so far.
Prat-Rojo, C., Pouille, P. A., Buceta, J. and Martin-Blanco, E. (2019). Mechanical coordination is sufficient to promote tissue replacement during metamorphosis in Drosophila. Embo J: e103594. PubMed ID: 31858605
During development, cells coordinate to organize in coherent structures. Although it is now well established that physical forces are essential for implementing this coordination, the instructive roles of mechanical inputs are not clear. This study shows that the replacement of the larval epithelia by the adult one in Drosophila demands the coordinated exchange of mechanical signals between two cell types, the histoblasts (adult precursors) organized in nests and the surrounding larval epidermal cells (LECs). An increasing stress gradient develops from the center of the nests toward the LECs as a result of the forces generated by histoblasts as they proliferate and by the LECs as they delaminate (push/pull coordination). This asymmetric radial coordination of expansive and contractile activities contributes to epithelial replacement. These analyses support a model in which cell-cell mechanical communication is sufficient for the rearrangements that implement epithelial morphogenesis.
Cho, B., Song, S. and Axelrod, J. D. (2020). Prickle isoforms determine handedness of helical morphogenesis. Elife 9. PubMed ID: 31934858
Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. This paper describes a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. The Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, it was shown that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. Evidence is provided that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pk(pk) or Pk(sple) predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes.

Thursday, February 20th, Signal Transduction

Banerjee, A. and Percival-Smith, A. (2020). Post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced. PLoS One 15(1): e0227642. PubMed ID: 31931520
Homeotic selector (HOX) transcription factors (TFs) regulate gene expression that determines the identity of Drosophila segments along the anterior-posterior (A-P) axis. The current challenge with HOX proteins is understanding how they achieve their functional specificity while sharing a highly conserved homeodomain (HD) that recognize the same DNA binding sites. One mechanism proposed to regulate HOX activity is differential post-translational modification (PTM). As a first step in investigating this hypothesis, the sites of PTM on a Sex combs reduced protein fused to a triple tag (SCRTT) extracted from developing embryos were identified by Tandem Mass Spectrometry (MS/MS). The PTMs identified include phosphorylation at S185, S201, T315, S316, T317 and T324, acetylation at K218, S223, S227, K309, K434 and K439, formylation at K218, K309, K325, K341, K369, K434 and K439, methylation at S19, S166, K168 and T364, carboxylation at D108, K298, W307, K309, E323, K325 and K369, and hydroxylation at P22, Y87, P107, D108, D111, P269, P306, R310, N321, K325, Y334, R366, P392 and Y398. Of the 44 modifications, 18 map to functionally important regions of SCR. Besides a highly conserved DNA-binding HD, HOX proteins also have functionally important, evolutionarily conserved small motifs, which may be Short Linear Motifs (SLiMs). SLiMs are proposed to be preferential sites of phosphorylation. Although 6 of 7 phosphosites map to regions of predicted SLiMs, no support was found for the hypothesis that the individual S, T and Y residues of predicted SLiMs are phosphorylated more frequently than S, T and Y residues outside of predicted SLiMs.
Chen, H. L., Kasuya, J., Lansdon, P., Kaas, G., Tang, H., Sodders, M. and Kitamoto, T. (2020). Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Drosophila Expressing Mutant Voltage-Gated Sodium Channels. G3 (Bethesda). PubMed ID: 32054635
Voltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, a forward genetic screen was conducted for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu. This analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, these results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.
Kaur, H., Sharma, S. K., Mandal, S. and Mandal, L. (2019). Lar maintains the homeostasis of the hematopoietic organ in Drosophila by regulating insulin signaling in the niche. Development 146(24). PubMed ID: 31784462
Stem cell compartments in metazoa get regulated by systemic factors as well as local stem cell niche-derived factors. However, the mechanisms by which systemic signals integrate with local factors in maintaining tissue homeostasis remain unclear. Employing the Drosophila lymph gland, which harbors differentiated blood cells, and stem-like progenitor cells and their niche, this study demonstrated how a systemic signal interacts and harmonizes with local factor/s to achieve cell type-specific tissue homeostasis. Genetic analyses uncovered a novel function of Lar, a receptor protein tyrosine phosphatase. Niche-specific loss of Lar leads to upregulated insulin signaling, causing increased niche cell proliferation and ectopic progenitor differentiation. Insulin signaling assayed by PI3K activation is downregulated after the second instar larval stage, a time point that coincides with the appearance of Lar in the hematopoietic niche. It was further demonstrated that Lar physically associates with InR and serves as a negative regulator for insulin signaling in the Drosophila larval hematopoietic niche. Whether Lar serves as a localized invariable negative regulator of systemic signals such as insulin in other stem cell niches remains to be explored.
Patel, A. L., Yeung, E., McGuire, S. E., Wu, A. Y., Toettcher, J. E., Burdine, R. D. and Shvartsman, S. Y. (2019). Optimizing photoswitchable MEK. Proc Natl Acad Sci U S A 116(51): 25756-25763. PubMed ID: 31796593
Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. This study presents evidence that psMEK indeed has suboptimal functionality in vivo, and a strategy is proposed to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, it was demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. It was establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, it esd udrf to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.
Kenwrick, K., Mukherjee, A. and Renault, A. D. (2019). Hmgcr promotes a long-range signal to attract Drosophila germ cells independently of Hedgehog. J Cell Sci 132(23). PubMed ID: 31719159
During development, many cell types migrate along stereotyped routes determined through deployment of cell surface or secreted guidance molecules. Although the identity of many of these molecules is known, the distances over which they natively operate can be difficult to determine. This study has quantified the range of an attractive signal for the migration of Drosophila germ cells. Their migration is guided by an attractive signal generated by the expression of genes in the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr) pathway, and by a repulsive signal generated by the expression of Wunens. This study demonstrates that the attractive signal downstream of Hmgcr is cell-contact independent and acts at long range, the extent of which depends on Hmgcr levels. This range would be sufficient to reach all of the germ cells for their entire migration. Furthermore, Hmgcr-mediated attraction does not require Wunens but can operate simultaneously with Wunen-mediated repulsion. Finally, several papers posit Hedgehog (Hh) as being the germ cell attractant downstream of Hmgcr. This study provides evidence that this is not the case.
Okada, H., Yagi, R., Gardeux, V., Deplancke, B. and Hafen, E. (2019). Sex-dependent and sex-independent regulatory systems of size variation in natural populations. Mol Syst Biol 15(11): e9012. PubMed ID: 31777173
Size of organs/organisms is a polygenic trait. Many of the growth-regulatory genes constitute conserved growth signaling pathways. However, how these multiple genes are orchestrated at the systems level to attain the natural variation in size including sexual size dimorphism is mostly unknown. This study has taken a multi-layered systems omics approach to study size variation in the Drosophila wing. Expression levels of many critical growth regulators such as Wnt and TGFbeta pathway components were shown to significantly differ between sexes but not between lines exhibiting size differences within each sex, suggesting a primary role of these regulators in sexual size dimorphism. Only a few growth genes including a receptor of steroid hormone ecdysone exhibit association with between-line size differences. In contrast, between-line size variation was found to be largely regulated by genes with a diverse range of cellular functions, most of which have never been implicated in growth. In addition, it was shown that expression quantitative trait loci (eQTLs) linked to these novel growth regulators accurately predict population-wide, between-line wing size variation. In summary, this study unveils differential gene regulatory systems that control wing size variation between and within sexes.

Wednesday, February 19th - Synapse and Vesicles

Pazos Obregon, F., Palazzo, M., Soto, P., Guerberoff, G., Yankilevich, P. and Cantera, R. (2019). An improved catalogue of putative synaptic genes defined exclusively by temporal transcription profiles through an ensemble machine learning approach. BMC Genomics 20(1): 1011. PubMed ID: 31870293
Assembly and function of neuronal synapses require the coordinated expression of a yet undetermined set of genes. Previously, an ensemble machine learning model was trained to assign a probability of having synaptic function to every protein-coding gene in Drosophila melanogaster. This approach resulted in the publication of a catalogue of 893 genes which was postulated to be very enriched in genes with a still undocumented synaptic function. Since then, the scientific community has experimentally identified 79 new synaptic genes. This study used these new empirical data to evaluate the original prediction. A series of changes were implemented to the training scheme of this model, and using the new data it was demonstrated that this improves its predictive power. Finally, the new synaptic genes were added to the training set and a new model was trained, obtaining a new, enhanced catalogue of putative synaptic genes. This study presents this new catalogue and announces that a regularly updated version will be available online. This study shows that training an ensemble of machine learning classifiers solely with the whole-body temporal transcription profiles of known synaptic genes resulted in a catalogue with a significant enrichment in undiscovered synaptic genes. Using new empirical data provided by the scientific community, the original approach was validated, improving the model to obtained an arguably more precise prediction. This approach reduces the number of genes to be tested through hypothesis-driven experimentation and will facilitate understanding of neuronal function.
Berke, B., Le, L. and Keshishian, H. (2020). Target-dependent retrograde signaling mediates synaptic plasticity at the Drosophila neuromuscular junction. Dev Neurobiol. PubMed ID: 31950660
Neurons that innervate multiple targets often establish synapses with target-specific strengths, and local forms of synaptic plasticity. This study has examined the molecular-genetic mechanisms that allow a single Drosophila motoneuron, the ventral Common Exciter (vCE), to establish connections with target-specific properties at its various synaptic partners. By driving transgenes in a subset of vCE's targets, it was found that individual target cells are able to independently control the properties of vCE's innervating branch and synapses. This is achieved by means of a trans-synaptic growth factor secreted by the target cell. At the larval neuromuscular junction, postsynaptic glutamate receptor activity stimulates the release of the BMP4/5/6 homolog Glass bottom boat (Gbb). As larvae mature and motoneuron terminals grow, Gbb activates the R-Smad transcriptional regulator phosphorylated Mad (pMad) to facilitate presynaptic development.Manipulations affecting glutamate receptors or Gbb within subsets of target muscles led to local effects either specific to the manipulated muscle or by a limited gradient within the presynaptic branches. While presynaptic development depends on pMad transcriptional activity within the motoneuron nucleus, this study found that the Gbb growth factor may also act locally within presynaptic terminals. Local Gbb signaling and presynaptic pMad accumulation within boutons may therefore participate in a "synaptic tagging" mechanism, to influence synaptic growth and plasticity in Drosophila.
Latcheva, N. K., Delaney, T. L., Viveiros, J. M., Smith, R. A., Bernard, K. M., Harsin, B., Marenda, D. R. and Liebl, F. L. W. (2019). The CHD protein, Kismet, is important for the recycling of synaptic vesicles during endocytosis. Sci Rep 9(1): 19368. PubMed ID: 31852969
Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. This study shows that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and Endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.
Graves, H. K., Jangam, S., Tan, K. L., Pignata, A., Seto, E. S., Yamamoto, S. and Wangler, M. F. (2019). A Genetic Screen for Genes That Impact Peroxisomes in Drosophila Identifies Candidate Genes for Human Disease. G3 (Bethesda). PubMed ID: 31767637
Peroxisomes are subcellular organelles that are essential for proper function of eukaryotic cells. In addition to being the sites of a variety of oxidative reactions, they are crucial regulators of lipid metabolism. Peroxisome loss or dysfunction leads to multi-system diseases in humans that strongly affect the nervous system. In order to identify previously unidentified genes and mechanisms that impact peroxisomes, a genetic screen was conducted on a collection of lethal mutations on the X chromosome in Drosophila. Using the number, size and morphology of GFP tagged peroxisomes as a readout, a screen was carried out for mutations that altered peroxisomes based on clonal analysis and confocal microscopy. From this screen, eighteen genes were identified that cause increases in peroxisome number or altered morphology when mutated. The human homologs were examined of these genes, and they were found to be involved in a diverse array of cellular processes. Interestingly, the human homologs from the X-chromosome collection are under selective constraint in human populations and are good candidate genes particularly for dominant genetic disease. This in vivo screening approach for peroxisome defects allows identification of novel genes that impact peroxisomes in vivo in a multicellular organism and is a valuable platform to discover genes potentially involved in dominant disease that could affect peroxisomes.
Bowers, M. R. and Reist, N. E. (2020). The C2A domain of synaptotagmin is an essential component of the calcium sensor for synaptic transmission. PLoS One 15(2): e0228348. PubMed ID: 32032373
The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. This study shows that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, mutation of these two key C2A hydrophobic residues were found to almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, this study reports the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.
Espadas, J., Pendin, D., Bocanegra, R., Escalada, A., Misticoni, G., Trevisan, T., Velasco Del Olmo, A., Montagna, A., Bova, S., Ibarra, B., Kuzmin, P. I., Bashkirov, P. V., Shnyrova, A. V., Frolov, V. A. and Daga, A. (2019). Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon. Nat Commun 10(1): 5327. PubMed ID: 31757972
The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. This study finds that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

Tuesday, February 18th - RNA and Transposons

Bronkhorst, A. W., Vogels, R., Overheul, G. J., Pennings, B., Gausson-Dorey, V., Miesen, P. and van Rij, R. P. (2019). A DNA virus-encoded immune antagonist fully masks the potent antiviral activity of RNAi in Drosophila. Proc Natl Acad Sci U S A 116(48): 24296-24302. PubMed ID: 31712431
Coevolution of viruses and their hosts may lead to viral strategies to avoid, evade, or suppress antiviral immunity. An example is antiviral RNA interference (RNAi) in insects: the host RNAi machinery processes viral double-stranded RNA into small interfering RNAs (siRNAs) to suppress viral replication, whereas insect viruses encode suppressors of RNAi, many of which inhibit viral small interfering RNA (vsiRNA) production. Yet, many studies have analyzed viral RNAi suppressors in heterologous systems, due to the lack of experimental systems to manipulate the viral genome of interest, raising questions about in vivo functions of RNAi suppressors. To address this caveat, an RNAi suppressor-defective mutant was generated of invertebrate iridescent virus 6 (IIV6), a large DNA virus in which previous work identified the 340R protein as a suppressor of RNAi. Loss of 340R did not affect vsiRNA production, indicating that 340R binds siRNA duplexes to prevent RNA-induced silencing complex assembly. Indeed, vsiRNAs were not efficiently loaded into Argonaute 2 during wild-type IIV6 infection. Moreover, IIV6 induced a limited set of mature microRNAs in a 340R-dependent manner, most notably miR-305-3p, which was attributed to stabilization of the miR-305-5p:3p duplex by 340R. The IIV6 340R deletion mutant did not have a replication defect in cells, but was strongly attenuated in adult Drosophila. This in vivo replication defect was completely rescued in RNAi mutant flies, indicating that 340R is a bona fide RNAi suppressor, the absence of which uncovers a potent antiviral immune response that suppresses virus accumulation approximately 100-fold. Together, this work indicates that viral RNAi suppressors may completely mask antiviral immunity.
Bhogal, J. K., Kanaskie, J. M. and DiAngelo, J. R. (2020). The role of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb27C in regulating lipid storage in the Drosophila fat body. Biochem Biophys Res Commun. PubMed ID: 31982137
The storage of excess nutrients as triglycerides is essential for all organisms to survive when food is scarce; however, the mechanisms by which triglycerides are stored are not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing that are important in the regulation of triglyceride storage. This lab has identified a number of splicing factors important for regulating lipid metabolism; however, the full complement of splicing proteins involved in achieving metabolic homeostasis is unknown. Heterogeneous nuclear ribonucleoproteins (hnRNPs), RNA binding proteins that inhibit the splicing of introns by preventing the assembly of splicing complexes, have no established metabolic functions. To assess any metabolic functions of hnRNPs, the GAL4/UAS system was used to induce RNAi to six hnRNP's: hnRNP-K, rumpelstiltskin (rump), smooth (sm), Hrb27C (also referred to as Hrp48), Hrb98DE, and Hrb87F in the Drosophila fat body. Decreasing the levels of hnRNP-K and rump resulted in a decrease in triglyceride storage, whereas decreasing the levels of sm, Hrb27C, and Hrb98DE resulted in an increase in triglyceride storage. The excess triglyceride phenotype in Hrb27C-RNAi flies resulted from both an increase in the number of fat body cells and the amount of fat stored per cell. In addition, both the splicing of the beta-oxidation gene, CPT1, and the expression of the lipase brummer (bmm) was altered in flies with decreased Hrb27C, providing insight into the lipid storage phenotype in these flies. Together, these results suggest that the hnRNP family of splicing factors have varying metabolic functions and may act on specific metabolic genes to control their expression and processing.
Vo, H. D. L., Wahiduzzaman, Tindell, S. J., Zheng, J., Gao, M. and Arkov, A. L. (2019). Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development. Sci Rep 9(1): 19190. PubMed ID: 31844131
The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte's posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using super-resolution microscopy, this study showed that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, this study showed that Aubergine forms homo-oligomers and using all purified Tudor domains, it was demonstrated that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. These data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their "interaction hubs", and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules.
Angelova, M. T., Dimitrova, D. G., Da Silva, B., Marchand, V., Jacquier, C., Achour, C., Brazane, M., Goyenvalle, C., Bourguignon-Igel, V., Shehzada, S., Khouider, S., Lence, T., Guerineau, V., Roignant, J. Y., Antoniewski, C., Teysset, L., Bregeon, D., Motorin, Y., Schaefer, M. R. and Carre, C. (2020). tRNA 2'-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila. Nucleic Acids Res. PubMed ID: 31943105
2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. This study identified two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. These results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.
Srivastav, S. P., Rahman, R., Ma, Q., Pierre, J., Bandyopadhyay, S. and Lau, N. C. (2019). Har-P, a short P-element variant, weaponizes P-transposase to severely impair Drosophila development. Elife 8. PubMed ID: 31845649
Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition causes an ovarian atrophy syndrome in Drosophila called gonadal dysgenesis (GD). Harwich (Har) strains with P-elements cause severe GD in F1 daughters when Har fathers mate with mothers lacking P-element-piRNAs (i.e. ISO1 strain). To address the mystery of why Har induces severe GD, hybrid Drosophila with Har genomic fragments were bred into the ISO1 background to create HISR-D or HISR-N lines that still cause Dysgenesis or are Non-dysgenic, respectively. In these lines, a highly truncated P-element variant that was named 'Har-P' that was discovered as the most frequent de novo insertion. Although HISR-D lines still contain full-length P-elements, HISR-N lines lost functional P-transposase but retained Har-P's that when crossed back to P-transposase restores GD induction. Finally, P-element-piRNA-directed repression was uncovered on Har-P's transmitted paternally to suppress somatic transposition. The Drosophila short Har-P's and full-length P-elements relationship parallels the MITEs/DNA-transposase in plants and SINEs/LINEs in mammals.
Amourda, C. and Saunders, T. E. (2020). The mirtron miR-1010 functions in concert with its host gene SKIP to balance elevation of nAcRbeta2. Sci Rep 10(1): 1688. PubMed ID: 32015391
Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. This study found that Drosophila larvae lacking either the mirtron miR-1010 or its binding site in the nicotinic acetylcholine receptor beta2 (nAcRbeta2) 3'UTR fail to grow properly and pupariate. Increase of cortical nAcRbeta2 mediated by neural activity elevates the level of intracellular Ca(2+), which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. miR-1010 downregulates nAcRbeta2. Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcRbeta2 mRNA levels (negative feedback loop). These results demonstrate how a mirtron, in coordination with its host gene, contributes to maintaining appropriate receptor levels, which in turn may play a role in maintaining homeostasis.

Friday, February 14th - Embryonic development

Rahimi, N., Averbukh, I., Carmon, S., Schejter, E. D., Barkai, N. and Shilo, B. Z. (2019). Dynamics of Spaetzle morphogen shuttling in the Drosophila embryo shapes gastrulation patterning. Development 146(21). PubMed ID: 31719046
Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, was followed in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. It is concluded that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.
Lim, J. and Choe, C. P. (2019). Functional analysis of engrailed in Tribolium segmentation. Mech Dev: 103594. PubMed ID: 31778794
The segment-polarity gene engrailed is required for segmentation in the early Drosophila embryo. Loss of Engrailed function results in segmentation defects that vary in severity from pair-rule phenotypes to a lawn phenotype lacking in obvious of segmentation. During segmentation, Engrailed is expressed in stripes with a single segmental periodicity in Drosophila, which is conserved in all arthropods examined so far. To define segments, the segmental stripes of Engrailed induce the segmental stripes of wingless at each parasegmental boundary. However, segmentation functions of orthologs of engrailed in non-Drosophila arthropods have yet to be reported. This study analyzed functions of the Tribolium ortholog of engrailed (Tc-engrailed) during embryonic segmentation. Larval cuticles with Tc-engrailed being knocked down had segmentation phenotypes including incomplete segment formation and loss of a group of segments. In agreement with the cuticle segmentation defects, segments developed incompletely and irregularly or did not form in Tribolium germbands where Tc-engrailed was knocked down. Furthermore, knock-down of Tc-engrailed did not properly express the segmental stripes of wingless in Tribolium germbands. Taken together with the conserved expression patterns of Engrailed in arthropod segmentation, these data suggest that Tc-engrailed is required for embryonic segmentation in Tribolium, and the genetic mechanism of Engrailed inducing wingless expression is conserved at least between Drosophila and Tribolium.
Huang, A., Rupprecht, J. F. and Saunders, T. E. (2020). Embryonic geometry underlies phenotypic variation in decanalized conditions. Elife 9. PubMed ID: 32048988
During development, many mutations cause increased variation in phenotypic outcomes, a phenomenon termed decanalization. Phenotypic discordance is often observed in the absence of genetic and environmental variations, but the mechanisms underlying such inter-individual phenotypic discordance remain elusive. Using the anterior-posterior (AP) patterning of the Drosophila embryo, embryonic geometry was identified as a key factor predetermining patterning outcomes under decanalizing mutations. With the wild-type AP patterning network, it was found that AP patterning is robust to variations in embryonic geometry; segmentation gene expression remains reproducible even when the embryo aspect ratio is artificially reduced by more than twofold. In contrast, embryonic geometry is highly predictive of individual patterning defects under decanalized conditions of either increased bicoid (bcd) dosage or bcd knockout. The phenotypic discordance can be traced back to variations in the gap gene expression, which is rendered sensitive to the geometry of the embryo under mutations.
Yoon, Y., Klomp, J., Martin-Martin, I., Criscione, F., Calvo, E., Ribeiro, J. and Schmidt-Ott, U. (2019). Embryo polarity in moth flies and mosquitoes relies on distinct old genes with localized transcript isoforms. Elife 8. PubMed ID: 31591963
Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. This study shows that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles.
Chatterjee, A., Aavula, K. and Nongthomba, U. (2019). Beadex, a homologue of the vertebrate LIM domain only protein, is a novel regulator of crystal cell development in Drosophila melanogaster. J Genet 98. PubMed ID: 31819023
Haematopoiesis is a complex process in which the regulatory mechanisms of several implicated transcription factors remain uncertain. Drosophila melanogaster is an excellent model to resolve the unanswered questions about the blood cell development. This study describes the role of Beadex, a Drosophila homologue of LIM domain only 2 (LMO2), in haematopoiesis. Mutants of Beadex were analysed for blood cell abnormalities. Crystal cells, a subset of haemocytes, were significantly more in Beadex hypermorphic flies. Similarly, Beadex misexpression in prohemocytes altered the crystal cell numbers. Stage-specific misexpression analyses demonstrated that Beadex functions after the prohemocytes enter the crystal cell lineage. It was also discovered that Pannier-U-shaped complex is a negative regulator of the crystal cell differentiation and is possibly negatively regulated by Beadex through its interaction with Pannier. This study therefore suggests the mechanism of two novel regulators of crystal cell specification-Beadex and Pannier-during Drosophila haematopoiesis.
Szikora, S., Gajdos, T., Novak, T., Farkas, D., Foldi, I., Lenart, P., Erdelyi, M. and Mihaly, J. (2020). Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes. J Cell Biol 219(1). PubMed ID: 31816054
Sarcomeres are extremely highly ordered macromolecular assemblies where structural organization is intimately linked to their functionality as contractile units. Although the structural basis of actin and Myosin interaction is revealed at a quasiatomic resolution, much less is known about the molecular organization of the I-band and H-zone. This study reports the development of a powerful nanoscopic approach, combined with a structure-averaging algorithm, that allowed determination of the position of 27 sarcomeric proteins in Drosophila melanogaster flight muscles with a quasimolecular, approximately 5- to 10-nm localization precision. With this protein localization atlas and template-based protein structure modeling, refined I-band and H-zone models were assembled with unparalleled scope and resolution. In addition, it was found that actin regulatory proteins of the H-zone are organized into two distinct layers, suggesting that the major place of thin filament assembly is an M-line-centered narrow domain where short actin oligomers can form and subsequently anneal to the pointed end.

Thursday, February 13th - Chromatin

Lepesant, J. M. J., Iampietro, C., Galeota, E., Auge, B., Aguirrenbengoa, M., Merce, C., Chaubet, C., Rocher, V., Haenlin, M., Waltzer, L., Pelizzola, M. and Di Stefano, L. (2019). A dual role of dLsd1 in oogenesis: regulating developmental genes and repressing transposons. Nucleic Acids Res. PubMed ID: 31799607
The histone demethylase LSD1 is a key chromatin regulator that is often deregulated in cancer. Its ortholog, dLsd1 plays a crucial role in Drosophila oogenesis; however, knowledge of dLsd1 function is insufficient to explain its role in the ovary. Genome-wide analysis was performed of dLsd1 binding in the ovary, and this paper documents that dLsd1 is preferentially associated to the transcription start site of developmental genes. An unanticipated interplay was uncovered between dLsd1 and the GATA transcription factor Serpent and an unexpected role is reported for Serpent in oogenesis. Besides, the transcriptomic data show that reducing dLsd1 levels results in ectopic transposable elements (TE) expression correlated with changes in H3K4me2 and H3K9me2 at TE loci. In addition, the results suggest that dLsd1 is required for Piwi dependent TE silencing. Hence, it is proposed that dLsd1 plays crucial roles in establishing specific gene expression programs and in repressing transposons during oogenesis.
Liu, M., Saha, N., Gajan, A., Saadat, N., Gupta, S. V. and Pile, L. A. (2019). A complex interplay between SAM synthetase and the epigenetic regulator SIN3 controls metabolism and transcription. J Biol Chem. PubMed ID: 31776190
The SIN3 histone-modifying complex regulates the expression of multiple methionine catabolic genes, including SAM synthetase (Sam-S), as well as S-adenosyl-methionine (SAM) levels. To further dissect the relationship between methionine catabolism and epigenetic regulation by SIN3, this study sought to identify genes and metabolic pathways controlled by SIN3 and SAM-S in Drosophila melanogaster. Using several approaches, including RNAi-mediated gene silencing, RNA-seq- and quantitative RT-PCR-based transcriptomics, and ultra-high performance LC-MS/MS- and GC/MS- based metabolomics, this study found that as a global transcriptional regulator, SIN3 impacted a wide range of genes and pathways. In contrast, SAM-S affected only a narrow range of genes and pathways. The expression and levels of additional genes and metabolites, however, were altered in Sin3A+Sam-S dual knockdown cells. This analysis revealed that SIN3 and SAM-S regulate overlapping pathways, many of which involve one-carbon and central carbon metabolisms. In some cases, the factors acted independently; in some others, redundantly; and for a third set, in opposition. Together, these results obtained from experiments with the chromatin regulator SIN3 and the metabolic enzyme SAM-S, uncover a complex relationship between metabolism and epigenetic regulation.
Varga, J., Korbai, S., Neller, A., Zsindely, N. and Bodai, L. (2019). Hat1 acetylates histone H4 and modulates the transcriptional program in Drosophila embryogenesis. Sci Rep 9(1): 17973. PubMed ID: 31784689
Post-translational modifications of histone proteins play a pivotal role in DNA packaging and regulation of genome functions. Histone acetyltransferase 1 (Hat1) proteins are conserved enzymes that modify histones by acetylating lysine residues. Hat1 is implicated in chromatin assembly and DNA repair but its role in cell functions is not clearly elucidated. We report the generation and characterization of a Hat1 loss-of-function mutant in Drosophila. Hat1 mutants are viable and fertile with a mild sub-lethal phenotype showing that Hat1 is not essential in fruit flies. Lack of Hat1 results in the near complete loss of histone H4 lysine (K) 5 and K12 acetylation in embryos, indicating that Hat1 is the main acetyltransferase specific for these marks in this developmental stage. Hat1 function and the presence of these acetyl marks are not required for the nuclear transport of histone H4 as histone variant His4r retained its nuclear localization both in Hat1 mutants and in His4r-K5R-K12R double point mutants. RNA-seq analysis of embryos indicate that in Hat1 mutants over 2000 genes are dysregulated and the observed transcriptional changes imply a delay in the developmental program of gene expression (Varga, 2019).
Chen, X., Ke, Y., Wu, K., Zhao, H., Sun, Y., Gao, L., Liu, Z., Zhang, J., Tao, W., Hou, Z., Liu, H., Liu, J. and Chen, Z. J. (2019). Key role for CTCF in establishing chromatin structure in human embryos. Nature 576(7786): 306-310. PubMed ID: 31801998
In the interphase of the cell cycle, chromatin is arranged in a hierarchical structure within the nucleus, which has an important role in regulating gene expression. However, the dynamics of 3D chromatin structure during human embryogenesis remains unknown. This study reports that, unlike mouse sperm, human sperm cells do not express the chromatin regulator CTCF and their chromatin does not contain topologically associating domains (TADs). Following human fertilization, TAD structure is gradually established during embryonic development. In addition, A/B compartmentalization is lost in human embryos at the 2-cell stage and is re-established during embryogenesis. Notably, blocking zygotic genome activation (ZGA) can inhibit TAD establishment in human embryos but not in mouse or Drosophila. Of note, CTCF is expressed at very low levels before ZGA, and is then highly expressed at the ZGA stage when TADs are observed. TAD organization is significantly reduced in CTCF knockdown embryos, suggesting that TAD establishment during ZGA in human embryos requires CTCF expression. These results indicate that CTCF has a key role in the establishment of 3D chromatin structure during human embryogenesis.
Gozalo, A., Duke, A., Lan, Y., Pascual-Garcia, P., Talamas, J. A., Nguyen, S. C., Shah, P. P., Jain, R., Joyce, E. F. and Capelson, M. (2019). Core Components of the Nuclear Pore Bind Distinct States of Chromatin and Contribute to Polycomb Repression. Mol Cell. PubMed ID: 31784359
Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. This study reports high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. This investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, this study identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. These findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93.
Palladino, J., Chavan, A., Sposato, A., Mason, T. D. and Mellone, B. G. (2020). Targeted De Novo Centromere Formation in Drosophila Reveals Plasticity and Maintenance Potential of CENP-A Chromatin. Dev Cell 52(3): 379-394. PubMed ID: 32049040
Centromeres are essential for accurate chromosome segregation and are marked by centromere protein A (CENP-A) nucleosomes. Mis-targeted CENP-A chromatin has been shown to seed centromeres at non-centromeric DNA. However, the requirements for such de novo centromere formation and transmission in vivo remain unknown. This study employed Drosophila melanogaster and the LacI/lacO system to investigate the ability of targeted de novo centromeres to assemble and be inherited through development. De novo centromeres form efficiently at six distinct genomic locations, which include actively transcribed chromatin and heterochromatin, and cause widespread chromosomal instability. During tethering, de novo centromeres sometimes prevail, causing the loss of the endogenous centromere via DNA breaks and HP1-dependent epigenetic inactivation. Transient induction of de novo centromeres and chromosome healing in early embryogenesis show that, once established, these centromeres can be maintained through development. These results underpin the ability of CENP-A chromatin to establish and sustain mitotic centromere function in Drosophila.

Wednesday, February 12th - Behavior

Zhang, W., Reeves, R. G. and Tautz, D. (2019). Identification of a genetic network for an ecologically relevant behavioral phenotype in Drosophila melanogaster. Mol Ecol. PubMed ID: 31867742
Pupation site choice of Drosophila third-instar larvae is critical for the survival of individuals, as pupae are exposed to various biotic and abiotic dangers while immobilized during the 3-4 days of metamorphosis. This singular behavioural choice is sensitive to both environmental and genetic factors. This study developed a high-throughput phenotyping approach to assay the variation in pupation height in Drosophila melanogaster, while controlling for possibly confounding factors. Substantial variation was found of mean pupation height among sampled natural stocks, and it was shown that the Drosophila Genetic Reference Panel (DGRP) reflects this variation. Using the DGRP stocks for genome wide association (GWA) mapping, 16 loci involved in determining pupation height could be resolved. The candidate genes in these loci are enriched for high expression in the larval central nervous system. A genetic network could be constructed from the candidate loci, which places scribble (scrib) at the centre, plus other genes known to be involved in nervous system development, such as Epidermal growth factor receptor (Egfr) and p53. Using gene disruption lines, it was possible to functionally validate several of the initially identified loci, as well as additional loci predicted from network analysis. This study shows that the combination of high throughput phenotyping with a genetic analysis of variation captured from the wild can be used to approach the genetic dissection of an environmentally relevant behavioural phenotype.
Pischedda, A., Shahandeh, M. P. and Turner, T. L. (2019). The loci of behavioral evolution: evidence that Fas2 and tilB underlie differences in pupation site choice behavior between Drosophila melanogaster and D. simulans. Mol Biol Evol. PubMed ID: 31774527
The behaviors of closely related species can be remarkably different, and these differences have important ecological and evolutionary consequences. While the recent boom in genotype-phenotype studies has led to a greater understanding of the genetic architecture and evolution of a variety of traits, studies identifying the genetic basis of behaviors are, comparatively, still lacking. This is likely because they are complex and environmentally sensitive phenotypes, making them difficult to measure reliably for association studies. The Drosophila species complex holds promise for addressing these challenges, as the behaviors of closely related species can be readily assayed in a common environment. This study investigated the genetic basis of an evolved behavioral difference, pupation site choice, between Drosophila melanogaster and D. simulans. In this study, A significant contribution was demonstrated of the X chromosome to the difference in pupation site choice behavior between these species. Using a panel of X-chromosome deficiencies, the majority of the X chromosome was screened for causal loci, and two regions were identified associated with this X-effect. Gene disruption and RNAi data were collecting supporting a single gene that affects pupation behavior within each region: Fas2 and tilB. Finally, differences in tilB expression were shown to correlate with the differences in pupation site choice behavior between species. This evidence associating two genes with differences in a complex, environmentally sensitive behavior represents the first step towards a functional and evolutionary understanding of this behavioral divergence.
Calhoun, A. J., Pillow, J. W. and Murthy, M. (2019). Unsupervised identification of the internal states that shape natural behavior. Nat Neurosci 22(12): 2040-2049. PubMed ID: 31768056
Internal states shape stimulus responses and decision-making, but methods to identify them are lacking. To address this gap, an unsupervised method was developed to identify internal states from behavioral data, and it was applied to a dynamic social interaction. During courtship, Drosophila melanogaster males pattern their songs using feedback cues from their partner. The model uncovers three latent states underlying this behavior and is able to predict moment-to-moment variation in song-patterning decisions. These states correspond to different sensorimotor strategies, each of which is characterized by different mappings from feedback cues to song modes. A pair of neurons previously thought to be command neurons for song production are sufficient to drive switching between states. These results reveal how animals compose behavior from previously unidentified internal states, which is a necessary step for quantitative descriptions of animal behavior that link environmental cues, internal needs, neuronal activity and motor outputs.
Zarin, A. A., Mark, B., Cardona, A., Litwin-Kumar, A. and Doe, C. Q. (2019). A multilayer circuit architecture for the generation of distinct locomotor behaviors in Drosophila. Elife 8. PubMed ID: 31868582
Animals generate diverse motor behaviors, yet how the same motor neurons (MNs) generate two distinct or antagonistic behaviors remains an open question. This study has characterized Drosophila larval muscle activity patterns and premotor/motor circuits to understand how they generate forward and backward locomotion. All body wall MNs were shown to be activated during both behaviors, but a subset of MNs change recruitment timing for each behavior. TEM was used to reconstruct a full segment of all 60 MNs and 236 premotor neurons (PMNs), including differentially-recruited MNs. Analysis of this comprehensive connectome identified PMN-MN 'labeled line' connectivity; PMN-MN combinatorial connectivity; asymmetric neuronal morphology; and PMN-MN circuit motifs that could all contribute to generating distinct behaviors. A recurrent network model was generated that reproduced the observed behaviors, and functional optogenetics was used to validate selected model predictions. This PMN-MN connectome will provide a foundation for analyzing the full suite of larval behaviors.
Greppi, C., Laursen, W. J., Budelli, G., Chang, E. C., Daniels, A. M., van Giesen, L., Smidler, A. L., Catteruccia, F. and Garrity, P. A. (2020). Mosquito heat seeking is driven by an ancestral cooling receptor. Science 367(6478): 681-684. PubMed ID: 32029627
Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. This study identified ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae. Although Ir21a mediates heat avoidance in Drosophila, this study found that it drives heat seeking and heat-stimulated blood feeding in Anopheles. At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. The data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera.
Shiozaki, H. M., Ohta, K. and Kazama, H. (2020). A Multi-regional Network Encoding Heading and Steering Maneuvers in Drosophila. Neuron. PubMed ID: 32023429
An internal sense of heading direction is computed from various cues, including steering maneuvers of the animal. Although neurons encoding heading and steering have been found in multiple brain regions, it is unclear whether and how they are organized into neural circuits. This study shows that, in flying Drosophila, heading and turning behaviors are encoded by population dynamics of specific cell types connecting the subregions of the central complex (CX), a brain structure implicated in navigation. Columnar neurons in the fan-shaped body (FB) of the CX exhibit circular dynamics that multiplex information about turning behavior and heading. These dynamics are coordinated with those in the ellipsoid body, another CX subregion containing a heading representation, although only FB neurons flip turn preference depending on the visual environment. Thus, the navigational system spans multiple subregions of the CX, where specific cell types show coordinated but distinct context-dependent dynamics.

Tuesday, February 11th - Adult physiology

Vasquez-Procopio, J., Osorio, B., Cortes-Martinez, L., Hernandez-Hernandez, F., Medina-Contreras, O., Rios-Castro, E., Comjean, A., Li, F., Hu, Y., Mohr, S., Perrimon, N. and Missirlis, F. (2019). Intestinal response to dietary manganese depletion in Drosophila. Metallomics. PubMed ID: 31799578
Manganese is considered essential for animal growth. Manganese ions serve as cofactors to three mitochondrial enzymes: superoxide dismutase (Sod2), arginase and glutamine synthase, and to glycosyltransferases residing in the Golgi. In Drosophila melanogaster, manganese has also been implicated in the formation of ceramide phosphoethanolamine, the insect's sphingomyelin analogue, a structural component of cellular membranes. Manganese overload leads to neurodegeneration and toxicity in both humans and Drosophila. This study reports specific absorption and accumulation of manganese during the first week of adulthood in flies that correlates with an increase in Sod2 activity during the same period. To test the requirement of dietary manganese for this accumulation, a Drosophila model of manganese deficiency was generated. Due to the lack of manganese-specific chelators, chemically defined media was used to grow the flies and deplete them of the metal. Dietary manganese depletion reduced Sod2 activity. Gene and protein expression changes were examined in the intestines of manganese depleted flies. Adaptive responses were found to the presumed loss of known manganese-dependent enzymatic activities: less glutamine synthase activity (amination of glutamate to glutamine) was compensated by 50% reduction in glutaminase (deamination of glutamine to glutamate); less glycosyltransferase activity, predicted to reduce protein glycosylation, was compensated by 30% reduction in lysosomal mannosidases (protein deglycosylating enzymes); less ceramide phosphoethanolamine synthase activity was compensated by 30% reduction in the Drosophila sphingomyeline phospodiesterase, which could catabolize ceramide phosphoethanolamine in flies. Reduced Sod2 activity, predicted to cause superoxide-dependent iron-sulphur cluster damage, resulted in cellular iron misregulation.
Lee, D. C., et al. (2019). Dietary supplementation with the ketogenic diet metabolite beta-hydroxybutyrate ameliorates post-TBI aggression in young-adult male Drosophila. Front Neurosci 13: 1140. PubMed ID: 31736687
Traumatic brain injury (TBI), caused by repeated concussive head trauma can induce chronic traumatic encephalopathy (CTE), a neurodegenerative disease featuring behavioral symptoms ranging from cognitive deficits to elevated aggression. In a Drosophila model, a high-impact trauma device is used to induce TBI-like symptoms and to study post-TBI behavioral outcomes. Following TBI, aggression in banged male flies was significantly elevated as compared with that in unbanged flies. These increases in aggressive behavior were not the result of basal motility changes, as measured by a negative geotaxis assay. Various forms of dietary therapy, especially the high-fat, low-carbohydrate ketogenic diet (KD), have recently been explored for a wide variety of neuropathies. It was thus hypothesized that putatively neuroprotective dietary interventions might be able to suppress post-traumatic elevations in aggressive behavior in animals subjected to head-trauma-inducing strikes, or "bangs". A normal high-carbohydrate Drosophila diet was supplemented with the KD metabolite beta-hydroxybutyrate (beta-HB)-a ketone body (KB). Banged flies raised on a KB-supplemented diet exhibited a marked reduction in aggression, whereas aggression in unbanged flies was equivalent whether dieted with KB supplements or not. Pharmacological blockade of the ATP-sensitive potassium (KATP) channel abrogated KB effects reducing post-TBI aggression while pharmacological activation mimicked them, suggesting a mechanism by which KBs act in this model. KBs did not significantly extend lifespan in banged flies, but markedly extended lifespan in unbanged flies. This study has thus developed a functional model for the study of post-TBI elevations of aggression. Further, it is concluded that dietary interventions may be a fruitful avenue for further exploration of treatments for TBI- and CTE-related cognitive-behavioral symptoms.
Schlame, M., Xu, Y., Erdjument-Bromage, H., Neubert, T. A. and Ren, M. (2020). Lipidome-wide (13)C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures. J Lipid Res 61(1): 95-104. PubMed ID: 31712250
Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, because it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are often used as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. A simple method to determine the turnover rate of phospholipid and acylglycerol species has been developed based on the incorporation of (13)C6-glucose combined with LC-MS/MS. Adult Drosophila melanogaster were labeled with (13)C6-glucose that incorporates into the entire lipidome, kinetic parameters were derived from mass spectra, and effects were studied of deletion of CG6718, the fly homolog of the calcium-independent phospholipase A2beta, on lipid metabolism. Although (13)C6-glucose gave rise to a complex pattern of (13)C incorporation, it was possible to identify discrete isotopomers in which (13)C atoms were confined to the glycerol group. With these isotopomers, turnover rate constants, half-life times, and fluxes of the glycerol backbone of were calculated of multiple lipid species. To perform these calculations, the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, was estimated by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When this method was applied to D. melanogaster, a range of lipid half-lives from 2 to 200 days was found, tissue-specific fluxes of individual lipid species were demonstrated, and a novel function was identified of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.
Liu, Y., Bao, H., Wang, W. and Lim, H. Y. (2019). Cardiac Snail family of transcription factors directs systemic lipid metabolism in Drosophila. PLoS Genet 15(11): e1008487. PubMed ID: 31725726
Maintenance of normal lipid homeostasis is crucial to heart function. On the other hand, the heart is now recognized to serve an important role in regulating systemic lipid metabolism; however, the molecular basis remains unclear. This study identified the Drosophila Snail family of transcription factors (herein termed Sna TFs) as new mediators of the heart control of systemic lipid metabolism. Overexpression of Sna TF genes specifically in the heart promotes whole-body leanness whereas their knockdown in the heart promotes obesity. In addition, flies that are heterozygous for a snail deficiency chromosome also exhibit systemic obesity, and cardiac-specific overexpression of Sna substantially reverses systemic obesity in these flies. It was further shown that genetically manipulating Sna TF levels in the fat body and intestine do not affect systemic lipid levels. Mechanistically, it was found that flies bearing the overexpression or inhibition of Sna TFs in the postnatal heart exhibit only systemic lipid metabolic defects but not heart abnormalities. Cardiac-specific alterations of Sna TF levels also do not perturb cardiac morphology, viability, lipid metabolism or fly food intake. On the other hand, cardiac-specific manipulations of Sna TF levels alter lipogenesis and lipolysis gene expression, mitochondrial biogenesis and respiration, and lipid storage droplet 1 and 2 (Lsd-1 and Lsd-2) levels in the fat body. Together, these results reveal a novel and specific role of Sna TFs in the heart on systemic lipid homeostasis maintenance that is independent of cardiac development and function and involves the governance of triglyceride synthesis and breakdown, energy utilization, and lipid droplet dynamics in the fat body.
Lovejoy, P. C. and Fiumera, A. C. (2019). Effects of dual exposure to the herbicides Atrazine and Paraquat on adult climbing ability and longevity in Drosophila melanogaster. Insects 10(11). PubMed ID: 31717666
Anthropomorphic effects are changing the planet, and therefore, organisms are being exposed to many new biotic and abiotic stressors. Exposure to multiple stressors can affect organisms in ways that are different than the sum of their individual effects, and these interactions are often difficult to predict. Atrazine and paraquat are two of the most widely used herbicides in the United States, and are individually known to increase oxidative damage, affect dopaminergic functioning, reduce longevity, and alter motor ability in non-target organisms. This study measured the effects of individual and combined exposure to low doses of atrazine and paraquat on climbing ability and longevity of Drosophila melanogaster. Atrazine and paraquat interact to affect D. melanogaster climbing ability and longevity in different ways. Atrazine appeared to have a weak mitigative effect against the decrease in climbing ability caused by paraquat. In contrast, combined exposure to atrazine and paraquat had detrimental synergistic effects on female longevity. Overall, this study shows that atrazine and paraquat can interact and that it is important to measure several traits when assessing the consequences of exposure to multiple stressors. Future studies should continue to assess the impacts of stressor interactions on organisms, as many combinations have never been examined.
Huynh, N., Ou, Q., Cox, P., Lill, R. and King-Jones, K. (2019). Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET. Nat Commun 10(1): 5463. PubMed ID: 31784520
Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. This study shows that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.

Monday, February 10th - Disease models

Belovich, A. N., Aguilar, J. I., Mabry, S. J., Cheng, M. H., Zanella, D., Hamilton, P. J., Stanislowski, D. J., Shekar, A., Foster, J. D., Bahar, I., Matthies, H. J. G. and Galli, A. (2019). A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster. Mol Psychiatry. PubMed ID: 31796894
Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. This study developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). Previous work has shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. This study demonstrates that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. This study showed that PIP2 also interacts with intracellular loop 4 at R443. Further, R443 was shown to electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, it was determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. This inhibition is associated with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, this study showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. This study presents the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.
Dubey, T., Gorantla, N. V., Chandrashekara, K. T. and Chinnathambi, S. (2019). Photoexcited Toluidine Blue Inhibits Tau Aggregation in Alzheimer's Disease. ACS Omega 4(20): 18793-18802. PubMed ID: 31737841
The aggregates of microtubule-associated protein Tau are considered as a major hallmark of Alzheimer's disease. Tau aggregates accumulate intracellularly leading to neuronal toxicity. Numerous approaches have been targeted against Tau protein aggregation, which include application of synthetic and natural compounds. Toluidine blue is a basic dye of phenothiazine family, which on irradiation with a 630 nm light gets converted into a photoexcited form, leading to generation of singlet oxygen species. Methylene blue is the parent compound of toluidine blue, which has been reported to be potent against tauopathy. The present work studied the potency of toluidine blue and photoexcited toluidine blue against Tau aggregation. Biochemical and biophysical analyses using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ThS fluorescence, circular dichroism spectroscopy, and electron microscopy suggested that toluidine blue inhibited the aggregation of Tau in vitro. The photoexcited toluidine blue potentially dissolved the matured Tau fibrils, which indicated the disaggregation property of toluidine blue. The cell biology studies including the cytotoxicity assay and reactive oxygen species (ROS) production assay suggested toluidine blue to be a biocompatible dye as it reduced ROS levels and cell death. The photoexcited toluidine blue modulates the cytoskeleton network in cells, which was supported by immunofluorescence studies of neuronal cells. The studies in a UAS Tau E14 transgenic Drosophila model suggested that photoexcited toluidine blue was potent to restore the survival and memory deficits of Drosophila. The overall finding of these studies suggested toluidine blue to be a potent molecule in rescuing the Tau-mediated pathology by inhibiting its aggregation, reducing the cell death, and modulating the tubulin levels and behavioral characteristics of Drosophila. Thus, toluidine blue can be addressed as a potent molecule against Alzheimer's disease.
Cox, R. L., Hofley, C. M., Tatapudy, P., Patel, R. K., Dayani, Y., Betcher, M. and LaRocque, J. R. (2019). Functional conservation of RecQ helicase BLM between humans and Drosophila melanogaster. Sci Rep 9(1): 17527. PubMed ID: 31772289
RecQ helicases are a family of proteins involved in maintaining genome integrity with functions in DNA repair, recombination, and replication. The human RecQ helicase family consists of five helicases: BLM, WRN, RECQL, RECQL4, and RECQL5. Inherited mutations in RecQ helicases result in Bloom Syndrome (BLM mutation), Werner Syndrome (WRN mutation), Rothmund-Thomson Syndrome (RECQL4 mutation), and other genetic diseases, including cancer. The RecQ helicase family is evolutionarily conserved, as Drosophila melanogaster have three family members: DmBlm, DmRecQL4, and DmRecQL5 and DmWRNexo, which contains a conserved exonuclease domain. DmBlm has functional similarities to human BLM (hBLM) as mutants demonstrate increased sensitivity to ionizing radiation (IR) and a decrease in DNA double-strand break (DSB) repair. To determine the extent of functional conservation of RecQ helicases, hBLM was expressed in Drosophila using the GAL4 > UASp system to determine if GAL4 > UASp::hBLM can rescue DmBlm mutant sensitivity to IR. hBLM was able to rescue female DmBlm mutant sensitivity to IR, supporting functional conservation. This functional conservation is specific to BLM, as human GAL4 > UASp::RECQL was not able to rescue DmBlm mutant sensitivity to IR. These results demonstrate the conserved role of BLM in maintaining the genome while reinforcing the applicability of using Drosophila as a model system to study Bloom Syndrome.
Ibrahim, R. B., Yeh, S. Y., Lin, K. P., Ricardo, F., Yu, T. Y., Chan, C. C., Tsai, J. W. and Liu, Y. T. (2019). Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration. Cell Mol Life Sci. PubMed ID: 31728576
Transthyretin amyloidosis (ATTR) is a progressive life-threatening disease characterized by the deposition of transthyretin (TTR) amyloid fibrils. Several pathogenic variants have been shown to destabilize TTR tetramers, leading to aggregation of misfolded TTR fibrils. However, factors that underlie the differential age of disease onset amongst amyloidogenic TTR variants remain elusive. This study examined the biological properties of various TTR mutations and found that the cellular secretory pattern of the wild-type (WT) TTR was similar to those of the late-onset mutant (Ala97Ser, p. Ala117Ser), stable mutant (Thr119Met, p. Thr139Met), early-onset mutant (Val30Met, p. Val50Met), but not in the unstable mutant (Asp18Gly, p. Asp38Gly). Cytotoxicity assays revealed their toxicities in the order of Val30Met > Ala97Ser > WT v Thr119Met in neuroblastoma cells. Surprisingly, while early-onset amyloidogenic TTR monomers (M-TTRs) are retained by the endoplasmic reticulum quality control (ERQC), late-onset amyloidogenic M-TTRs can be secreted extracellularly. Treatment of thapsigargin (Tg) to activate the unfolded protein response (UPR) alleviates Ala97Ser M-TTR secretion. Interestingly, Ala97Ser TTR overexpression in Drosophila causes late-onset fast neurodegeneration and a relatively short lifespan, recapitulating human disease progression. This study demonstrates that the escape of TTR monomers from the ERQC may underlie late-onset amyloidogenesis in patients and suggests that targeting ERQC could mitigate late-onset ATTR.
Fang, E. F., Hou, Y., Lautrup, S., Jensen, M. B., Yang, B., SenGupta, T., Caponio, D., Khezri, R., Demarest, T. G., Aman, Y., Figueroa, D., Morevati, M., Lee, H. J., Kato, H., Kassahun, H., Lee, J. H., Filippelli, D., Okur, M. N., Mangerich, A., Croteau, D. L., Maezawa, Y., Lyssiotis, C. A., Tao, J., Yokote, K., Rusten, T. E., Mattson, M. P., Jasper, H., Nilsen, H. and Bohr, V. A. (2019). NAD(+) augmentation restores mitophagy and limits accelerated aging in Werner syndrome. Nat Commun 10(1): 5284. PubMed ID: 31754102
Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. This study reports impaired mitophagy and depletion of NAD(+), a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD(+) biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD(+) repletion restores NAD(+) metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD(+) repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. these findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD(+) levels counteracts WS phenotypes.
Imai, Y., Inoshita, T., Meng, H., Shiba-Fukushima, K., Hara, K. Y., Sawamura, N. and Hattori, N. (2019). Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson's disease. Commun Biol 2: 424. PubMed ID: 31799427
Mitochondrial degeneration is considered one of the major causes of Parkinson's disease (PD). Improved mitochondrial functions are expected to be a promising therapeutic strategy for PD. This study introduced a light-driven proton transporter, Delta-rhodopsin (dR), to Drosophila mitochondria, where the mitochondrial proton-motive force (Deltap) and mitochondrial membrane potential are maintained in a light-dependent manner. The loss of the PD-associated mitochondrial gene CHCHD2 resulted in reduced ATP production, enhanced mitochondrial peroxide production and lower Ca(2+)-buffering activity in dopaminergic (DA) terminals in flies. These cellular defects were improved by the light-dependent activation of mitochondrion-targeted dR (mito-dR). Moreover, mito-dR reversed the pathology caused by the CHCHD2 deficiency to suppress alpha-synuclein aggregation, DA neuronal loss, and elevated lipid peroxidation in brain tissue, improving motor behaviors. This study suggests the enhancement of Deltap by mito-dR as a therapeutic mechanism that ameliorates neurodegeneration by protecting mitochondrial functions.

Friday, February 7th - Adult neural development and function

Sancer, G., Kind, E., Uhlhorn, J., Volkmann, J., Hammacher, J., Pham, T., Plazaola-Sasieta, H. and Wernet, M. F. (2019). Cellular and synaptic adaptations of neural circuits processing skylight polarization in the fly. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 31811399
Specialized ommatidia harboring polarization-sensitive photoreceptors exist in the 'dorsal rim area' (DRA) of virtually all insects. Although downstream elements have been described both anatomically and physiologically throughout the optic lobes and the central brain of different species, little is known about their cellular and synaptic adaptations and how these shape their functional role in polarization vision. Previous work has shown that in the DRA of Drosophila melanogaster, two distinct types of modality-specific 'distal medulla' cell types (Dm-DRA1 and Dm-DRA2) are post-synaptic to long visual fiber photoreceptors R7 and R8, respectively. This study describes additional neuronal elements in the medulla neuropil that manifest modality-specific differences in the DRA region, including DRA-specific neuronal morphology, as well as differences in the structure of pre- or post-synaptic membranes. Furthermore, certain cell types (medulla tangential cells and octopaminergic neuromodulatory cells) were shown to specifically avoid contacts with polarization-sensitive photoreceptors. Finally, while certain transmedullary cells are specifically absent from DRA medulla columns, other subtypes show specific wiring differences while still connecting the DRA to the lobula complex, as has previously been described in larger insects. This hints towards a complex circuit architecture with more than one pathway connecting polarization-sensitive DRA photoreceptors with the central brain.
Marchetti, G. and Tavosanis, G. (2019). Modulators of hormonal response regulate temporal fate specification in the Drosophila brain. PLoS Genet 15(12): e1008491. PubMed ID: 31809495
How a progenitor sequentially produces neurons of different fates and the impact of extrinsic signals conveying information about developmental progress or environmental conditions on this process represent key, but elusive questions. Each of the four progenitors of the Drosophila mushroom body (MB) sequentially gives rise to the MB neuron subtypes. The temporal fate determination pattern of MB neurons can be influenced by extrinsic cues, conveyed by the steroid hormone ecdysone. This study shows that the activation of Transforming Growth Factor-beta (TGF-beta) signalling via glial-derived Myoglianin regulates the fate transition between the early-born alpha'beta' and the pioneer alphabeta MB neurons by promoting the expression of the ecdysone receptor B1 isoform (EcR-B1). While TGF-beta signalling is required in MB neuronal progenitors to promote the expression of EcR-B1, ecdysone signalling acts postmitotically to consolidate the alpha'beta' MB fate. Indeed, it is proposed that if these signalling cascades are impaired alpha'beta' neurons lose their fate and convert to pioneer alphabeta. Conversely, an intrinsic signal conducted by the zinc finger transcription factor Kruppel-homolog 1 (Kr-h1) antagonises TGF-beta signalling and acts as negative regulator of the response mediated by ecdysone in promoting alpha'beta' MB neuron fate consolidation. Taken together, the consolidation of alpha'beta' MB neuron fate requires the response of progenitors to local signalling to enable postmitotic neurons to sense a systemic signal.
Liu, X., Shen, J., Xie, L., Wei, Z., Wong, C., Li, Y., Zheng, X., Li, P. and Song, Y. (2019). Mitotic implantation of the transcription factor Prospero via phase separation drives terminal neuronal differentiation. Dev Cell. PubMed ID: 31866201
Compacted heterochromatin blocks are prevalent in differentiated cells and present a barrier to cellular reprogramming. It remains obscure how heterochromatin remodeling is orchestrated during cell differentiation. This study found that the evolutionarily conserved homeodomain transcription factor Prospero (Pros)/Prox1 ensures neuronal differentiation by driving heterochromatin domain condensation and expansion. Intriguingly, in mitotically dividing Drosophila neural precursors, Pros is retained at H3K9me3(+) pericentromeric heterochromatin regions of chromosomes via liquid-liquid phase separation (LLPS). During mitotic exit of neural precursors, mitotically retained Pros recruits and concentrates heterochromatin protein 1 (HP1) into phase-separated condensates and drives heterochromatin compaction. This establishes a transcriptionally repressive chromatin environment that guarantees cell-cycle exit and terminal neuronal differentiation. Importantly, mammalian Prox1 employs a similar "mitotic-implantation-ensured heterochromatin condensation" strategy to reinforce neuronal differentiation. Together, these results unveiled a new paradigm whereby mitotic implantation of a transcription factor via LLPS remodels H3K9me3(+) heterochromatin and drives timely and irreversible terminal differentiation.
Liu, Z., Chen, Y. and Rao, Y. (2020). An RNAi screen for secreted factors and cell-surface players in coordinating neuron and glia development in Drosophila. Mol Brain 13(1): 1. PubMed ID: 31900209
The establishment of the functional nervous system requires coordinated development of neurons and glia in the embryo. Our understanding of underlying molecular and cellular mechanisms, however, remains limited. The developing Drosophila visual system is an excellent model for understanding the developmental control of the nervous system. By performing a systematic transgenic RNAi screen, this study investigated the requirements of secreted proteins and cell-surface receptors for the development of photoreceptor neurons (R cells) and wrapping glia (WG) in the Drosophila visual system. From the screen, w seven genes whose knockdown disrupted the development of R cells and/or WG were identified, including amalgam (ama), domeless (dome), epidermal growth factor receptor (EGFR), kuzbanian (kuz), N-Cadherin (CadN), neuroglian (nrg), and shotgun (shg). Cell-type-specific analysis revealed that ama is required in the developing eye disc for promoting cell proliferation and differentiation, which is essential for the migration of glia in the optic stalk. These results also suggest that nrg functions in both eye disc and WG for coordinating R-cell and WG development.
Howard, C. E., Chen, C. L., Tabachnik, T., Hormigo, R., Ramdya, P. and Mann, R. S. (2019). Serotonergic Modulation of Walking in Drosophila. Curr Biol 29(24): 4218-4230.e4218. PubMed ID: 31786064
To navigate complex environments, animals must generate highly robust, yet flexible, locomotor behaviors. For example, walking speed must be tailored to the needs of a particular environment. Not only must animals choose the correct speed and gait, they must also adapt to changing conditions and quickly respond to sudden and surprising new stimuli. Neuromodulators, particularly the small biogenic amine neurotransmitters, have the ability to rapidly alter the functional outputs of motor circuits. This study shows that the serotonergic system in the vinegar fly, Drosophila melanogaster, can modulate walking speed in a variety of contexts and also change how flies respond to sudden changes in the environment. These multifaceted roles of serotonin in locomotion are differentially mediated by a family of serotonergic receptors with distinct activities and expression patterns.
Pena, C. D., Zhang, S., Markey, M., Venkatesh, T. and Vazquez, M. (2019). Collective behaviors of Drosophila-derived retinal progenitors in controlled microenvironments. PLoS One 14(12): e0226250. PubMed ID: 31835272
Collective behaviors of retinal progenitor cells (RPCs) are critical to the development of neural networks needed for vision. The collective migration of heterogeneous groups of RPCs in response to dynamic signaling fields of development remains incompletely understood. This project used a controlled, microfluidic assay to produce dynamic signaling fields of Fibroblast Growth Factor (FGF) that stimulated the chemotactic migration of primary RPCs extracted from Drosophila. Results illustrated collective RPC chemotaxis dependent on average size of clustered cells, in contrast to the non-directional movement of individually-motile RPCs. Quantitative study of these diverse collective responses will advance understanding of retina developmental processes, and aid study/treatment of inherited eye disease. Lastly, this unique coupling of defined invertebrate models with tunable microfluidic assays provides advantages for future quantitative and mechanistic study of varied RPC migratory responses.

Thursday, February 6th - Signaling

Sheard, K. M., Thibault-Sennett, S. A., Sen, A., Shewmaker, F. and Cox, R. T. (2019). Clueless forms dynamic, insulin-responsive bliss particles sensitive to stress. Dev Biol. PubMed ID: 31837288
Drosophila Clueless (Clu) is a ribonucleoprotein that directly affects mitochondrial function. Loss of clu causes mitochondrial damage, and Clu associates with proteins on the mitochondrial outer membrane. Clu's subcellular pattern is diffuse throughout the cytoplasm, but Clu also forms large mitochondria-associated particles. Clu particles are reminiscent of ribonucleoprotein particles such as stress granules and processing bodies. Ribonucleoprotein particles play critical roles in the cell by regulating mRNAs spatially and temporally. This study shows that Clu particles are unique, highly dynamic and rapidly disperse in response to stress in contrast to processing bodies and autophagosomes. In addition, Clu particle formation is dependent on diet as ovaries from starved females no longer contain Clu particles, and insulin signaling is necessary and sufficient for Clu particle formation. Oxidative stress also disperses particles. Since Clu particles are only present under optimal conditions,they have been termed "bliss particles". It was also demonstrated that many aspects of Clu function are conserved in the yeast homolog Clu1p. These observations identify Clu particles as stress-sensitive cytoplasmic particles whose absence corresponds with altered cell stress and mitochondrial localization.
Das, P., et al. (2019). Maternal almondex, a neurogenic gene, is required for proper subcellular Notch distribution in early Drosophila embryogenesis. Dev Growth Differ. PubMed ID: 31782145
Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. This study examined the role of almondex in Notch signaling during early Drosophila embryogenesis. In addition to being required for lateral inhibition in the neuroectoderm, almondex was also found to be partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, it was found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, it is speculated that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.
Beckmann, J. F., Sharma, G. D., Mendez, L., Chen, H. and Hochstrasser, M. (2019). The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors. Elife 8. PubMed ID: 31774393
Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where both host effects and high-copy suppressors of toxicity were identified. The strongest suppressor was karyopherin-alpha, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-alpha; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-alpha, P32, or CidA in female flies suppressed CI. It is proposed that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.
Gao, Y., Zhang, X., Xiao, L., Zhai, C., Yi, T., Wang, G., Wang, E., Ji, X., Hu, L., Shen, G. and Wu, S. (2019). Usp10 Modulates the Hippo Pathway by Deubiquitinating and Stabilizing the Transcriptional Coactivator Yorkie. Int J Mol Sci 20(23). PubMed ID: 31795326
The Hippo signaling pathway is an evolutionarily conserved regulator that plays important roles in organ size control, homeostasis, and tumorigenesis. As the key effector of the Hippo pathway, Yorkie (Yki) binds to transcription factor Scalloped (Sd) and promotes the expression of target genes, leading to cell proliferation and inhibition of apoptosis. Thus, it is of great significance to understand the regulatory mechanism for Yki protein turnover. This study provides evidence that the deubiquitinating enzyme ubiquitin-specific protease 10 (Usp10) binds Yki to counteract Yki ubiquitination and stabilize Yki protein in Drosophila S2 cells. The results in Drosophila wing discs indicate that silence of Usp10 decreases the transcription of target genes of the Hippo pathway by reducing Yki protein. In vivo functional analysis ulteriorly showed that Usp10 upregulates the Yki activity in Drosophila eyes. These findings uncover Usp10 as a novel Hippo pathway modulator and provide a new insight into the regulation of Yki protein stability and activity.
Tsuda-Sakurai, K., Kimura, M. and Miura, M. (2019). Diphthamide modification of eEF2 is required for gut tumor-like hyperplasia induced by oncogenic Ras. Genes Cells. PubMed ID: 31828897
Eukaryotic elongation factor 2 (eEF2) undergoes a unique post-translational modification called diphthamidation. Although eEF2 diphthamidation is highly conserved, its pathophysiological function is still largely unknown. To elucidate the function of diphthamidation in tumor, this study examined the involvement of diphthamidation pathway enzyme Dph5 in tumor progression in Drosophila adult gut. Expression of oncogenic Ras(V12) in gut intestinal stem cells (ISCs) and enteroblasts (EBs) causes hypertrophy and disruption of gut epithelia, and shortened lifespan. Knockdown of Dph5 ameliorated these pathogenic phenotypes. Dph5 is required for gross translation activation and high dMyc protein level in Ras(V12) tumor-like hyperplasia. Transcriptome analysis revealed that Dph5 is involved in the regulation of ribosome biogenesis genes. These results suggest that diphthamidation is required for translation activation partly through the regulation of ribosome biogenesis in Ras-induced tumor-like hyperplasia model in Drosophila gut.
Tettweiler, G., Blaquiere, J. A., Wray, N. B. and Verheyen, E. M. (2019). Hipk is required for JAK/STAT activity during development and tumorigenesis. PLoS One 14(12): e0226856. PubMed ID: 31891940
Drosophila has been instrumental as a model system in studying signal transduction and revealing molecular functions in development and human diseases. A point mutation in the Drosophila Janus kinase JAK (called hop) causes constitutive activation of the JAK/STAT pathway. This study provides robust genetic evidence that the Homeodomain interacting protein kinase (Hipk) is required for endogenous JAK/STAT activity. Overexpression of Hipk can phenocopy the effects of overactive JAK/STAT mutations and lead to melanized tumors, and loss of Hipk can suppress the effects of hyperactive JAK/STAT. Further, the loss of the pathway effector Stat92E can suppress Hipk induced overgrowth. Interaction studies show that Hipk can physically interact with Stat92E and regulate Stat92E subcellular localization. Together the results show that Hipk is a novel factor required for effective JAK/STAT signaling.

Wednesday, February 5th - Cytoskeleton and Junctions

Caldwell, J. T., Mermelstein, D. J., Walker, R. C., Bernstein, S. I. and Huxford, T. (2019). X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties. J Mol Biol. PubMed ID: 31786266
Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. This study reports the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using a system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain were prepared and crystallized. The x-ray crystal structure was solved by molecular replacement, and the resulting model was refined against diffraction data to 2.2 A resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.
Finegan, T. M., Hervieux, N., Nestor-Bergmann, A., Fletcher, A. G., Blanchard, G. B. and Sanson, B. (2019). The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension. PLoS Biol 17(12): e3000522. PubMed ID: 31805038
In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less is known. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions (AJs). A previous study discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well-known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of AJs. This study surveyed a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular AJs (tAJs), the first of its kind. Clonal analysis showed that two cells, rather than three cells, contributing Sdk are sufficient for tAJ localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where AJs are actively remodelled, the phenotype of sdk null mutant embryos during Drosophila axis extension was analyzed using quantitative methods. Apical cell shapes were found to be abnormal in sdk mutants, suggesting a defect in tissue remodelling during convergence and extension. Moreover, adhesion at apical vertices is compromised in rearranging cells, with apical tears in the cortex forming and persisting throughout axis extension, especially at the centres of rosettes. Finally, it was shown that polarised cell intercalation is decreased in sdk mutants. Mathematical modelling of the cell behaviours supports the notion that the T1 transitions of polarised cell intercalation (referring to the rearrangement of groups of four cells) are delayed in sdk mutants, in particular in rosettes. It is proposed that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the abnormal apical cell shapes in sdk mutant embryos.
Zheng, S., West, J. J., Yu, C. G. and Harris, T. J. C. (2019). Arf-GEF localization and function at myosin-rich adherens junctions via coiled-coil hetero-dimerization with an adaptor protein. Mol Biol Cell: mbcE19100566. PubMed ID: 31693432
Tissue dynamics require regulated interactions between adherens junctions and cytoskeletal networks. For example, myosin-rich adherens junctions recruit the cytohesin Arf-GEF Steppke, which down-regulates junctional tension and facilitates tissue stretching. This recruitment mechanism was dissected with structure-function and other analyses of Steppke and Stepping stone, an implicated adaptor protein. During Drosophila dorsal closure, Steppke's coiled-coil domain was necessary and sufficient for junctional recruitment. Purified coiled-coil domains of Steppke and Stepping stone hetero-dimerized through a hydrophobic surface of the Steppke domain. This mapped surface was required for Steppke's junctional localization and tissue regulation. Stepping stone co-localized with Steppke at junctions, and was required for junctional Steppke localization and proper tissue stretching. A second conserved region of Stepping stone was necessary and largely sufficient for junctional localization. Remarkably, this region could substitute for the Steppke coiled-coil domain for junction localization and regulation, suggesting the main role of the Steppke coiled-coil domain is linkage to the junctional targeting region of Stepping stone. Thus, coiled-coil hetero-dimerization with Stepping stone normally recruits Step to junctions. Intriguingly, Stepping stone's junctional localization also seems partly dependent on Steppke.
Diaz, U., Bergman, Z. J., Johnson, B. M., Edington, A. R., de Cruz, M. A., Marshall, W. F. and Riggs, B. (2019). Microtubules are necessary for proper Reticulon localization during mitosis. PLoS One 14(12): e0226327. PubMed ID: 31877164
During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. This study examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, this study shows that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, it was shown that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.
Ahmed, Z., Mazumdar, S. and Ray, K. (2019). Kinesin associated protein, DmKAP, binding harnesses the C-terminal ends of the Drosophila kinesin-2 stalk heterodimer. Biochem Biophys Res Commun. PubMed ID: 31784087
The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Uusing the Fluorescence Lifetime assay this study showed that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2alpha stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.
Aguilar-Aragon, M., Fletcher, G. and Thompson, B. J. (2019). The cytoskeletal motor proteins Dynein and MyoV direct apical transport of Crumbs. Dev Biol. PubMed ID: 31881198
Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. This study used the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded- whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.

Tuesday, February 4th - Adult Physiology

Camus, M. F., O'Leary, M., Reuter, M. and Lane, N. (2020). Impact of mitonuclear interactions on life-history responses to diet. Philos Trans R Soc Lond B Biol Sci 375(1790): 20190416. PubMed ID: 31787037
Mitochondria are central to both energy metabolism and biosynthesis. Mitochondrial function could therefore influence resource allocation. Critically, mitochondrial function depends on interactions between proteins encoded by the mitochondrial and nuclear genomes. Severe incompatibilities between these genomes can have pervasive effects on both fitness and longevity. How milder deficits in mitochondrial function affect life-history trade-offs is less well understood. This study analysed how mitonuclear interactions affect the trade-off between fecundity and longevity in Drosophila melanogaster. A panel of 10 different mitochondrial DNA haplotypes are considered against two contrasting nuclear backgrounds (w(1118) (WE) and Zim53 (ZIM)) in response to high-protein versus standard diet.Strikingly different responses are reported between the two nuclear backgrounds. WE females have higher fecundity and decreased longevity on high protein. ZIM females have much greater fecundity and shorter lifespan than WE flies on standard diet. High protein doubled their fecundity with no effect on longevity. Mitochondrial haplotype reflected nuclear life-history trade-offs, with a negative correlation between longevity and fecundity in WE flies and no correlation in ZIM flies. Mitonuclear interactions had substantial effects but did not reflect genetic distance between mitochondrial haplotypes. It is concluded that mitonuclear interactions can have significant impact on life-history trade-offs, but their effects are not predictable by relatedness. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.
Chiang, A. C., McCartney, E., O'Farrell, P. H. and Ma, H. (2019). A genome-wide screen reveals that reducing mitochondrial DNA polymerase can promote elimination of deleterious mitochondrial mutations. Curr Biol 29(24): 4330-4336.e4333. PubMed ID: 31786061
A mutant mitochondrial genome arising amid the pool of mitochondrial genomes within a cell must compete with existing genomes to survive to the next generation. Even weak selective forces can bias transmission of one genome over another to affect the inheritance of mitochondrial diseases and guide the evolution of mitochondrial DNA (mtDNA). Studies in several systems suggested that purifying selection in the female germline reduces transmission of detrimental mitochondrial mutations. In contrast, some selfish genomes can take over despite a cost to host fitness. Within individuals, the outcome of competition is therefore influenced by multiple selective forces. The nuclear genome, which encodes most proteins within mitochondria, and all external regulators of mitochondrial biogenesis and dynamics can influence the competition between mitochondrial genomes, yet little is known about how this works. Previous work established a Drosophila line transmitting two mitochondrial genomes in a stable ratio enforced by purifying selection benefiting one genome and a selfish advantage favoring the other. In this study, to find nuclear genes that impact mtDNA competition, heterozygous deletions were screened tiling approximately 70% of the euchromatic regions, and their influence on this ratio was examined. This genome-wide screen detected many nuclear modifiers of this ratio and identified one as the catalytic subunit of mtDNA polymerase gene (POLG), tam. A reduced dose of tam drove elimination of defective mitochondrial genomes. This study suggests that this approach will uncover targets for interventions that would block propagation of pathogenic mitochondrial mutations.
Wei, M., Shi, L., Kong, R., Zhao, H. and Li, Z. (2019). Heparan sulfate maintains adult midgut homeostasis in Drosophila. Cell Biol Int. PubMed ID: 31868274
Tissue homeostasis is controlled by differentiated progeny of residential progenitors (stem cells). Adult stem cells constantly adjust their proliferation/differentiation rates to respond to tissue damage and stresses. However, how differentiated cells maintain tissue homeostasis remains unclear. This study found that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, protects differentiated cells from loss to maintain intestinal homeostasis. HS depletion in enterocytes (ECs) leads to intestinal homeostasis disruption, with accumulation of intestinal stem cell (ISC)-like cells and mis-differentiated progeny. HS-deficient ECs are prone to cell death/stress and induced cytokine and epidermal growth factor (EGF) expression, which in turn promote ISC proliferation and differentiation. Interestingly, HS depletion in ECs results in inactivation of Decapentaplegic (Dpp) signaling. Moreover, ectopic Dpp signaling completely rescued the defects caused by HS depletion. Together, these data demonstrate that HS is required for Dpp signal activation in ECs, thereby protecting ECs from ablation to maintain midgut homeostasis. These data shed light into the regulatory mechanisms of how differentiated cells contribute to tissue homeostasis maintenance.
Yang, X., Liang, J., Ding, L., Li, X., Lam, S. M., Shui, G., Ding, M. and Huang, X. (2019). Phosphatidylserine synthetase regulates cellular homeostasis through distinct metabolic mechanisms. PLoS Genet 15(12): e1008548. PubMed ID: 31869331
Phosphatidylserine (PS), synthesized in the endoplasmic reticulum (ER) by phosphatidylserine synthetase (PSS; CG4825), is transported to the plasma membrane (PM) and mitochondria through distinct routes. The in vivo functions of PS at different subcellular locations and the coordination between different PS transport routes are not fully understood. This paper reports that Drosophila PSS regulates cell growth, lipid storage and mitochondrial function. In pss RNAi, reduced PS depletes plasma membrane Akt, contributing to cell growth defects; the metabolic shift from phospholipid synthesis to neutral lipid synthesis results in ectopic lipid accumulation; and the reduction of mitochondrial PS impairs mitochondrial protein import and mitochondrial integrity. Importantly, reducing PS transport from the ER to PM by loss of PI4KIIIalpha partially rescues the mitochondrial defects of pss RNAi. Together, these results uncover a balance between different PS transport routes and reveal that PSS regulates cellular homeostasis through distinct metabolic mechanisms.
Wetzker, C. and Reinhardt, K. (2019). Distinct metabolic profiles in Drosophila sperm and somatic tissues revealed by two-photon NAD(P)H and FAD autofluorescence lifetime imaging. Sci Rep 9(1): 19534. PubMed ID: 31862926
Metabolic profiles vary across all levels of biological diversity, from cells to taxa. Two-photon fluorescence lifetime imaging microscopy (FLIM) facilitates metabolic characterisation of biological specimens by assaying the intrinsic autofluorescence of the ubiquitous coenzymes NAD(P)H and FAD. The potential of this method for characterising the diversity of organismal metabolism remains largely untapped. Using FLIM in Drosophila melanogaster, this study shows tissue-specificity in fluorescence lifetime that reflects variation in redox patterns. In particular, sperm cells exhibited elevated glycolysis relative to other tissues. Sperm metabolism was shown to be phenotypically plastic: compared to male-stored sperm, sperm stored in the female's storage organ showed a substantial reduction in the protein-bound FAD lifetime fraction but no change in the NAD(P)H profile. This study represents the first ex vivo investigation of sperm metabolism using FLIM.
Xie, J., Wang, D., Ling, S., Yang, G., Yang, Y. and Chen, W. (2019). High-salt diet causes sleep fragmentation in young Drosophila through circadian rhythm and dopaminergic Systems. Front Neurosci 13: 1271. PubMed ID: 31849585
Salt (sodium chloride) is an essential dietary requirement, but excessive consumption has long-term adverse consequences. A high-salt diet (HSD) increases the risk of chronic diseases such as cardiovascular conditions and diabetes and is also associated with poor sleep quality. Little is known, however, about the neural circuit mechanisms that mediate HSD-induced sleep changes. This study sought to identify the effects of HSD on the sleep and related neural circuit mechanisms of Drosophila. Strikingly, it was found that HSD causes young Drosophila to exhibit a fragmented sleep phenotype similar to that of normal aging individuals. Importantly, it was further shown that HSD slightly impairs circadian rhythms and that the HSD-induced sleep changes are dependent on the circadian rhythm system. In addition, it was demonstrated that HSD-induced sleep changes are dopaminergic-system dependent. Together, these results provide insight into how elevated salt in the diet can affect sleep quality.

Monday, February 3rd - Chromatin

Demakova, O. V., Demakov, S. A., Boldyreva, L. V., Zykova, T. Y., Levitsky, V. G., Semeshin, V. F., Pokholkova, G. V., Sidorenko, D. S., Goncharov, F. P., Belyaeva, E. S. and Zhimulev, I. F. (2019). Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes. Chromosoma. PubMed ID: 31820086
In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, four principle chromatin states (4capital EN, Cyrilliccapital EM, Cyrilliccapital EM, Cyrillic model) were uncovered in the fruit fly, and these were matched to the structures observed in polytene chromosomes. Ruby/malachite chromatin states form black bands containing developmental genes, whereas aquamarine chromatin corresponds to interbands enriched with 5' regions of ubiquitously expressed genes. Lazurite chromatin supposedly forms faint gray bands and encompasses the bodies of housekeeping genes. This report tests this idea using the X chromosome as the model and MSL1 as a protein marker of the lazurite chromatin. Bioinformatic analysis indicates that in the X chromosome, it is only the lazurite chromatin that is simultaneously enriched for the proteins and histone marks associated with exons, transcription elongation, and dosage compensation. As a result of FISH and EM mapping of a dosage compensation complex subunit, MSL1, this study provides direct evidence that lazurite chromatin forms faint gray bands. This analysis proves that overall most of housekeeping genes typically span from the interbands (5' region of the gene) to the gray band (gene body). More rarely, active lazurite chromatin and inactive malachite/ruby chromatin may be found within a common band, where both the housekeeping and the developmental genes reside together.
Miller, D. E. (2019). Synaptonemal complex-deficient Drosophila melanogaster females exhibit rare DSB repair events, recurrent copy-number variation, and an increased rate of de novo transposable element movement. G3 (Bethesda). PubMed ID: 31882405
Genetic stability depends on the maintenance of a variety of chromosome structures and the precise repair of DNA breaks. During meiosis, programmed double-strand breaks (DSBs) made in prophase I are normally repaired as gene conversions or crossovers. DSBs can also be made by other mechanisms, such as the movement of transposable elements (TEs), which must also be resolved. Incorrect repair of these DNA lesions can lead to mutations, copy-number changes, translocations, and/or aneuploid gametes. In Drosophila melanogaster, as in most organisms, meiotic DSB repair occurs in the presence of a rapidly evolving multiprotein structure called the synaptonemal complex (SC). This study used whole-genome sequencing to investigate the fate of meiotic DSBs in D. melanogaster mutant females lacking functional SC, to assay for de novo copy-number variation (CNV) formation, and to examine the role of the SC in transposable element movement in flies. The data indicate that, in the absence of SC, copy-number variation still occurs and meiotic DSB repair by gene conversion occurs infrequently. Remarkably, an 856-kilobase de novo CNV was observed in two unrelated individuals of different genetic backgrounds and was identical to a CNV recovered in a previous wild-type study, suggesting that recurrent formation of large CNVs occurs in Drosophila. In addition, the rate of novel TE insertion was markedly higher than wild type in one of two SC mutants tested, suggesting that SC proteins may contribute to the regulation of TE movement and insertion in the genome. Overall, this study provides novel insight into the role that the SC plays in genome stability and provides clues as to why the sequence, but not structure, of SC proteins is rapidly evolving.
Ueberschar, M., Wang, H., Zhang, C., Kondo, S., Aoki, T., Schedl, P., Lai, E. C., Wen, J. and Dai, Q. (2019). BEN-solo factors partition active chromatin to ensure proper gene activation in Drosophila. Nat Commun 10(1): 5700. PubMed ID: 31836703
The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein-Elba3-bridges Elba1 and Elba2 to form an ELBA complex. This study report comprehensive investigation of these proteins in Drosophila embryos. Common and distinct binding sites were assessed for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. It was further demonstrated that ELBA collaborates with other insulators to regulate developmental patterning. Finally, this study found that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.
Ellison, C. E. and Cao, W. (2019). Nanopore sequencing and Hi-C scaffolding provide insight into the evolutionary dynamics of transposable elements and piRNA production in wild strains of Drosophila melanogaster. Nucleic Acids Res. PubMed ID: 31754714
Illumina sequencing has allowed for population-level surveys of transposable element (TE) polymorphism via split alignment approaches, which has provided important insight into the population dynamics of TEs. However, such approaches are not able to identify insertions of uncharacterized TEs, nor can they assemble the full sequence of inserted elements. This study used nanopore sequencing and Hi-C scaffolding to produce de novo genome assemblies for two wild strains of Drosophila melanogaster from the Drosophila Genetic Reference Panel (DGRP). Ovarian piRNA populations and Illumina split-read TE insertion profiles have been previously produced for both strains. Nanopore sequencing with Hi-C scaffolding produces highly contiguous, chromosome-length scaffolds, and this study identified hundreds of TE insertions that were missed by Illumina-based methods, including a novel micropia-like element that has recently invaded the DGRP population. Hundreds of piRNA-producing loci were found that are specific to each strain. Some of these loci are created by strain-specific TE insertions, while others appear to be epigenetically controlled. These results suggest that Illumina approaches reveal only a portion of the repetitive sequence landscape of eukaryotic genomes and that population-level resequencing using long reads is likely to provide novel insight into the evolutionary dynamics of repetitive elements.
Melnikova, L., Molodina, V., Erokhin, M., Georgiev, P. and Golovnin, A. (2019). HIPP1 stabilizes the interaction between CP190 and Su(Hw) in the Drosophila insulator complex. Sci Rep 9(1): 19102. PubMed ID: 31836797
Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). This study performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720-892) and with CP190, but not with Mod(mdg4)-67.2. Hipp1 null mutants (Hipp1Delta1) were generated and the loss of Hipp1 did not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilizes the interaction between CP190 and the Su(Hw)-dependent complex.
Albig, C., Wang, C., Dann, G. P., Wojcik, F., Schauer, T., Krause, S., Maenner, S., Cai, W., Li, Y., Girton, J., Muir, T. W., Johansen, J., Johansen, K. M., Becker, P. B. and Regnard, C. (2019). JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila. Nat Commun 10(1): 5343. PubMed ID: 31767855
In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. This study shows that JIL-1's targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein; CG7946). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, interactors of the endogenous JJ-complex were identified, and it is proposed that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome.
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