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


Friday March 30th, 2018

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Cairns, L., Tran, T., Fowl, B. H., Patterson, A., Kim, Y. J., Bothner, B. and Kavran, J. M. (2018). Salvador has an extended SARAH domain that mediates binding to Hippo kinase. J Biol Chem. PubMed ID: 29519817
The Hippo pathway controls cell proliferation and differentiation through the precisely tuned activity of a core kinase cassette. The activity of Hippo kinase is modulated by interactions between its C-terminal coiled-coil, termed the SARAH domain, and the SARAH domains of either dRassF or Salvador. This study examined the molecular basis of SARAH domain mediated interactions and their influence on Hippo kinase activity. Focused was placed on Salvador, a positive effector of Hippo activity and the least well characterized SARAH domain-containing protein. The crystal structure was determined of a complex between Salvador and Hippo SARAH domains from Drosophila. This structure provided insight into the organization of the Salvador SARAH domain including a folded N-terminal extension that expands the binding interface with Hippo SARAH domain. This extension was found to improve the solubility of Salvador SARAH domain, enhances binding to Hippo, and is unique to Salvador. It is therefore suggested that the definition of the Salvador SARAH domain be expanded to include this extended region. The heterodimeric assembly observed in the crystal was confirmed by crosslinked mass spectrometry and provided a structural basis for the mutually exclusive interactions of Hippo with either dRassF or Salvador. Salvador influenced the kinase activity of Mst2, the mammalian Hippo homolog. In co-transfected HEK293T cells, human Salvador increased the levels of Mst2 autophosphorylation and Mst2-mediated phosphorylation of select substrates, whereas Salvador SARAH domain inhibited Mst2 autophosphorylation in vitro. These results suggest Salvador enhances the effects of Hippo kinase activity at multiple points in the Hippo pathway.
Campbell, K., Lebreton, G., Franch-Marro, X. and Casanova, J. (2018). Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth. PLoS Genet 14(2): e1007167. PubMed ID: 29420531
Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue this study has focused studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, this study recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. The differences between Snail and Serpent correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorkie target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a 'multi hit' effect may be achieved through the aberrant activation of just a single gene.
Cong, B., Ohsawa, S. and Igaki, T. (2018). JNK and Yorkie drive tumor progression by generating polyploid giant cells in Drosophila. Oncogene. PubMed ID: 29535423
Epithelial cancer tissues often possess polyploid giant cells, which are thought to be highly oncogenic. However, the mechanisms by which polyploid giant cells are generated in tumor tissues and how such cells contribute to tumor progression remain elusive. Cells mutant for the endocytic gene rab5 in Drosophila imaginal epithelium exhibit enlarged nuclei. This study finds that mutations in endocytic 'neoplastic tumor-suppressor' genes, such as rab5, vps25, erupted, or avalanche result in generation of polyploid giant cells. Genetic analyses on rab5-defective cells reveal that cooperative activation of JNK and Yorkie generates polyploid giant cells via endoreplication. Mechanistically, Yorkie-mediated upregulation of Diap1 cooperates with JNK to downregulate the G2/M cyclin CycB, thereby inducing endoreplication. Interestingly, malignant tumors induced by Ras activation and cell polarity defect also consist of polyploid giant cells, which are generated by JNK and Yorkie-mediated downregulation of CycB. Strikingly, elimination of polyploid giant cells from such malignant tumors by blocking endoreplication strongly suppressed tumor growth and metastatic behavior. These observations suggest that JNK and Yorkie, two oncogenic proteins activated in many types of human cancers, cooperatively drive tumor progression by generating oncogenic polyploid giant cells.
Bai, Z., Li, Z. and Xiao, W. (2018). Drosophila bendless catalyzes K63-linked polyubiquitination and is involved in the response to DNA damage. Mutat Res 808: 39-47. PubMed ID: 29518634
This study reports the identification and functional characterization of the Drosophila bendless/ubc13 gene, encoding a unique ubiquitin-conjugating enzyme (Ubc or E2), in DNA-damage response. Ben forms a heterodimer with DmUev1a, the only Ubc/E2 variant (Uev) in Drosophila. Ben and DmUev1a act together to catalyze K63-linked polyubiquitination in vitro. ben can functionally rescue the yeast ubc13 null mutant from killing by DNA-damaging agents. Ben(P97S), which was previously described to affect the connectivity between the giant fiber and the tergotrochanter motor neuron, fails to interact with the RING protein Chfr but retains interaction with DmUev1a as well as Uevs from other species. The corresponding yeast Ubc13P97S interacts with Mms2 but fails to bind Rad5. Consequently, neither benP97S nor ubc13P97S is able to complement the yeast ubc13 mutant defective in error-free DNA-damage tolerance. More importantly, the benP97S mutant flies are more sensitive to a DNA-damaging agent, suggesting that Ben functions in a manner similar to its yeast and mammalian counterparts. Collectively, these observations imply that Ben-DmUev1a-promoted K63-linked polyubiquitination and involvement in DNA-damage response are highly conserved in eukaryotes including flies.
Corcoran, J. A., Sonntag, Y., Andersson, M. N., Johanson, U. and Lofstedt, C. (2018). Endogenous insensitivity to the Orco agonist VUAA1 reveals novel olfactory receptor complex properties in the specialist fly Mayetiola destructor. Sci Rep 8(1): 3489. PubMed ID: 29472565
Insect olfactory receptors are routinely expressed in heterologous systems for functional characterisation. It was recently discovered that the essential olfactory receptor co-receptor (Orco) of the Hessian fly, Mayetiola destructor (Mdes), does not respond to the agonist VUAA1, which activates Orco in all other insects analysed to date. Using a mutagenesis-based approach this study identified three residues in MdesOrco, located in different transmembrane helices as supported by 3D modelling, that confer sensitivity to VUAA1. Reciprocal mutations in Drosophila melanogaster (Dmel) and the noctuid moth Agrotis segetum (Aseg) Orcos diminish sensitivity of these proteins to VUAA1. Additionally, mutating these residues in DmelOrco and AsegOrco compromised odourant receptor (OR) dependent ligand-induced Orco activation. In contrast, both wild-type and VUAA1-sensitive MdesOrco were capable of forming functional receptor complexes when coupled to ORs from all three species, suggesting unique complex properties in M. destructor, and that not all olfactory receptor complexes are "created" equal.
Chang, Y. C., Wu, J. W., Hsieh, Y. C., Huang, T. H., Liao, Z. M., Huang, Y. S., Mondo, J. A., Montell, D. and Jang, A. C. (2018). Rap1 negatively regulates the Hippo pathway to polarize directional protrusions in collective cell migration. Cell Rep 22(8): 2160-2175. PubMed ID: 29466741
In collective cell migration, directional protrusions orient cells in response to external cues, which requires coordinated polarity among the migrating cohort. However, the molecular mechanism has not been well defined. Drosophila border cells (BCs) migrate collectively and invade via the confined space between nurse cells, offering an in vivo model to examine how group polarity is organized. This study shows that the front/back polarity of BCs requires Rap1, hyperactivation of which disrupts cluster polarity and induces misoriented protrusions and loss of asymmetry in the actin network. Conversely, hypoactive Rap1 causes fewer protrusions and cluster spinning during migration. A forward genetic screen revealed that downregulation of the Hippo (Hpo) pathway core components hpo or mats enhances the Rap1V12-induced migration defect and misdirected protrusions. Mechanistically, association of Rap1V12 with the kinase domain of Hpo suppresses its activity, which releases Hpo signaling-mediated suppression of F-actin elongation, promoting cellular protrusions in collective cell migration.

Thursday, March 29th

Bohra, A. A., Kallman, B. R., Reichert, H. and VijayRaghavan, K. (2018). Identification of a single pair of interneurons for bitter taste processing in the Drosophila brain. Curr Biol 28(6): 847-858.e843. PubMed ID: 29502953
Drosophila has become an excellent model system for investigating the organization and function of the gustatory system due to the relatively simple neuroanatomical organization of its brain and the availability of powerful genetic and transgenic technology. Thus, at the molecular and cellular levels, a great deal of insight into the peripheral detection and coding of gustatory information has already been attained. In contrast, much less is known about the central neural circuits that process this information and induce behaviorally appropriate motor output. This study combined functional behavioral tests with targeted transgene expression through specific driver lines to identify a single bilaterally homologous pair of bitter-sensitive interneurons that are located in the subesophageal zone of the brain. Anatomical and functional data indicate that these interneurons receive specific synaptic input from bitter-sensitive gustatory receptor neurons. Targeted transgenic activation and inactivation experiments show that these bitter-sensitive interneurons can largely suppress the proboscis extension reflex to appetitive stimuli, such as sugar and water. These functional experiments, together with calcium-imaging studies and calcium-modulated photoactivatable ratiometric integrator (CaMPARI) labeling, indicate that these first-order local interneurons play an important role in the inhibition of the proboscis extension reflex that occurs in response to bitter tastants. Taken together, these studies present a cellular identification and functional characterization of a key gustatory interneuron in the bitter-sensitive gustatory circuitry of the adult fly.
Louis, T., Stahl, A., Boto, T. and Tomchik, S. M. (2018). Cyclic AMP-dependent plasticity underlies rapid changes in odor coding associated with reward learning. Proc Natl Acad Sci U S A 115(3): E448-e457. PubMed ID: 29284750
Learning and memory rely on dopamine and downstream cAMP-dependent plasticity across diverse organisms. Despite the central role of cAMP signaling, it is not known how cAMP-dependent plasticity drives coherent changes in neuronal physiology that encode the memory trace, or engram. In Drosophila, the mushroom body (MB) is critically involved in olfactory classical conditioning, and cAMP signaling molecules are necessary and sufficient for normal memory in intrinsic MB neurons. To evaluate the role of cAMP-dependent plasticity in learning, this study examined how cAMP manipulations and olfactory classical conditioning modulate olfactory responses in the MB with in vivo imaging. Elevating cAMP pharmacologically or optogenetically produced plasticity in MB neurons, altering their responses to odorants. Odor-evoked Ca(2+) responses showed net facilitation across anatomical regions. At the single-cell level, neurons exhibited heterogeneous responses to cAMP elevation, suggesting that cAMP drives plasticity to discrete subsets of MB neurons. Olfactory appetitive conditioning enhanced MB odor responses, mimicking the cAMP-dependent plasticity in directionality and magnitude. Elevating cAMP to equivalent levels as appetitive conditioning also produced plasticity, suggesting that the cAMP generated during conditioning affects odor-evoked responses in the MB. Finally, this plasticity was found to be dependent on the Rutabaga type I adenylyl cyclase, linking cAMP-dependent plasticity to behavioral modification. Overall, these data demonstrate that learning produces robust cAMP-dependent plasticity in intrinsic MB neurons, which is biased toward naturalistic reward learning. This suggests that cAMP signaling may serve to modulate intrinsic MB responses toward salient stimuli.
Berns, D. S., DeNardo, L. A., Pederick, D. T. and Luo, L. (2018). Teneurin-3 controls topographic circuit assembly in the hippocampus. Nature 554(7692): 328-333. PubMed ID: 29414938
Evolutionary Homolog Study
Brain functions rely on specific patterns of connectivity. Teneurins are evolutionarily conserved transmembrane proteins that instruct synaptic partner matching in Drosophila and are required for vertebrate visual system development. The roles of vertebrate teneurins in connectivity beyond the visual system remain largely unknown and their mechanisms of action have not been demonstrated. This study shows that mouse teneurin-3 (see Drosophila Tenascin major) is expressed in multiple topographically interconnected areas of the hippocampal region, including proximal CA1, distal subiculum, and medial entorhinal cortex. Viral-genetic analyses reveal that teneurin-3 is required in both CA1 and subicular neurons for the precise targeting of proximal CA1 axons to distal subiculum. Furthermore, teneurin-3 promotes homophilic adhesion in vitro in a splicing isoform-dependent manner. These findings demonstrate striking genetic heterogeneity across multiple hippocampal areas and suggest that teneurin-3 may orchestrate the assembly of a complex distributed circuit in the mammalian brain via matching expression and homophilic attraction.
Baggett, V., Mishra, A., Kehrer, A. L., Robinson, A. O., Shaw, P. and Zars, T. (2018). Place learning overrides innate behaviors in Drosophila. Learn Mem 25(3): 122-128. PubMed ID: 29449456
Animals in a natural environment confront many sensory cues. Some of these cues bias behavioral decisions independent of experience, and action selection can reveal a stimulus-response (S-R) connection. How animals use learning to modify S-R relationships is a largely open question. Three sensory stimuli, air, light, and gravity sources were presented to individual Drosophila melanogaster in both naive and place conditioning situations. Flies were tested for a potential modification of the S-R relationships of anemotaxis, phototaxis, and negative gravitaxis by a contingency that associated place with high temperature. With two stimuli, significant S-R relationships were abandoned when the cue was in conflict with the place learning contingency. The role of the dunce (dnc) cAMP-phosphodiesterase and the rutabaga (rut) adenylyl cyclase were examined in all conditions. Both dnc1 and rut2080 mutant flies failed to display significant S-R relationships with two attractive cues, and have characteristically lower conditioning scores under most conditions. Thus, learning can have profound effects on separate native S-R relationships in multiple contexts, and mutation of the dnc and rut genes reveal complex effects on behavior.
Zhao, X., Lenek, D., Dag, U., Dickson, B. and Keleman, K. (2018). Persistent activity in a recurrent circuit underlies courtship memory in Drosophila. Elife 7. PubMed ID: 29322941
Recurrent connections are thought to be a common feature of the neural circuits that encode memories, but how memories are laid down in such circuits is not fully understood. This study presents evidence that courtship memory in Drosophila relies on the recurrent circuit between mushroom body γ (MBγ), M6 output, and aSP13 dopaminergic neurons. Persistent neuronal activity of aSP13 neurons was demonstrated; it transiently potentiates synaptic transmission from MBγ>M6 neurons. M6 neurons in turn provide input to aSP13 neurons, prolonging potentiation of MBγ>M6 synapses over time periods that match short-term memory. These data support a model in which persistent aSP13 activity within a recurrent circuit lays the foundation for a short-term memory.
Schnaitmann, C., Haikala, V., Abraham, E., Oberhauser, V., Thestrup, T., Griesbeck, O. and Reiff, D. F. (2018). Color processing in the early visual system of Drosophila. Cell 172(1-2): 318-330.e318. PubMed ID: 29328919
Color vision is encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. This study examined the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. Color-opponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale" and "yellow" ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently.

Wednesday, March 28th

Yadav, A. K. and Srikrishna, S. (2018). scribble (scrib) knockdown induces tumorigenesis by modulating Drp1-Parkin mediated mitochondrial dynamics in the wing imaginal tissues of Drosophila. Mitochondrion [Epub ahead of print]. PubMed ID: 29360576
scrib loss of function is associated with various human-cancers. Most of the human-cancers have been characterized by mitochondrial dysfunction with elevated oxidative stress. However, the role of scrib to mitochondrial dysfunction in cancer has not been investigated earlier. This study has shown that scrib knockdown leads to mitochondrial depolarization, fragmentation and perinuclear-clustering along with disruption of the redox homeostasis. Moreover, the scrib abrogated tumor showed the elevation of Drp-1 and reduced expression of Marf, which suggests enhanced mitochondrial-fission. Further, the reduced expression of Parkin and HtrA2 interpret defective mitophagy leading to clustering of fragmented mitochondria and apoptotic inhibition in scrib knockdown tumors. Also, Parkin immunostaining depicted its reduced expression and mislocalization in the tumor's mitochondria in comparison to wild type. Moreover, the genetic study revealed the epistatic interactions of parkin and scrib. Thus, for the first time these results suggested that scrib loss induced mitochondrial-dysfunction modulates cancer progression by altering the mitochondrial dynamics regulators.
Zhang, Z., Cao, W. and Edery, I. (2018). The SR protein B52/SRp55 regulates splicing of the period thermosensitive intron and mid-day siesta in Drosophila. Sci Rep 8(1): 1872. PubMed ID: 29382842
Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as temperature increases, an adaptive response that aims to minimize the deleterious effects from exposure to heat. This temperature-dependent plasticity in mid-day sleep levels is partly based on the thermal sensitive splicing of an intron in the 3' untranslated region (UTR) of the circadian clock gene termed period (per). This study evaluated a possible role for the serine/arginine-rich (SR) splicing factors in the regulation of the 3’-terminal intron (termed dmpi8) from period splicing efficiency and mid-day siesta. Using a Drosophila cell culture assay B52/SRp55 increases dmpi8 splicing efficiency, whereas other SR proteins have little to no effect. The magnitude of the stimulatory effect of B52 on dmpi8 splicing efficiency is modulated by natural variation in single nucleotide polymorphisms (SNPs) in the per 3' UTR that correlate with B52 binding levels. Down-regulating B52 expression in clock neurons increases mid-day siesta and reduces dmpi8 splicing efficiency. These results establish a novel role for SR proteins in sleep and suggest that polymorphisms in the per 3' UTR contribute to natural variation in sleep behavior by modulating the binding efficiencies of SR proteins.
Ziegler, A. B., Maniere, G. and Grosjean, Y. (2018). JhI-21 plays a role in Drosophila insulin-like peptide release from larval IPCs via leucine transport. Sci Rep 8(1): 1908. PubMed ID: 29382949
Insulin is present all across the animal kingdom. Its proper release after feeding is of extraordinary importance for nutrient uptake, regulation of metabolism, and growth. This study used Drosophila melanogaster to shed light on the processes linking dietary leucine intake to insulin secretion. The Drosophila genome encodes 8 insulin-like peptides ('Dilps'). Of these, Dilp2 is secreted after the ingestion of a leucine-containing diet. Minidiscs, related to mammalian system-L transporters, acts as a leucine sensor within the Dilp2-secreting insulin-producing cells ("IPCs") of the brain. This study shows that a second leucine transporter, JhI-21, of the same family is additionally necessary for proper leucine sensing in the IPCs. Using calcium imaging and ex-vivo cultured brains this study shows that knockdown of JhI-21 in IPCs causes malfunction of these cells: they are no longer able to sense dietary leucine or to release Dilp2 in a leucine dependent manner. JhI-21 knockdown in IPCs further causes systemic metabolic defects including defective sugar uptake and altered growth. Finally, it was shown that JhI-21 and Minidiscs have no cumulative effect on Dilp2 release. Since system-L transporters are expressed by mammalian beta-cells these results could help to better understand the role of these proteins in insulin signaling.
Akai, N., Igaki, T. and Ohsawa, S. (2018). Wingless signaling regulates winner/loser status in Minute cell competition. Genes Cells 23(3): 234-240. PubMed ID: 29431244
Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild-type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, two assay systems were establised for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3-RNAi-expressing cells) system in which RpS3-RNAi-expressing wing pouch cells are eliminated from wild-type wing disc and (ii) the SURE (supercompetition of RpS3-expressing clones in RpS3/+ tissue) system in which RpS3-over-expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells. An ectopic over-expression screen using the CORE system identified Wg signaling as a critical regulator of Minute cell competition. Activation of Wg signaling in loser cells suppressed their elimination, whereas down-regulation of Wg signaling in loser cells enhanced their elimination. Furthermore, using the SURE system, it was found that down-regulation of Wg signaling in winner cells suppressed elimination of neighboring losers. These observations suggest that cellular Wg signaling activity is crucial for determining winner/loser status and thereby triggering Minute cell competition.
Aguilar-Aragon, M., Elbediwy, A., Foglizzo, V., Fletcher, G. C., Li, V. S. W. and Thompson, B. J. (2018). Pak1 kinase maintains apical membrane identity in epithelia. Cell Rep 22(7): 1639-1646. PubMed ID: 29444419
Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. This study shows that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. These results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia.
Aydogan, M. G., Wainman, A., Saurya, S., Steinacker, T. L., Caballe, A., Novak, Z. A., Baumbach, J., Muschalik, N. and Raff, J. W. (2018). A homeostatic clock sets daughter centriole size in flies. J Cell Biol. PubMed ID: 29500190
Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing 'mother' centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. This study shows that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size.

Tuesday, March 27th

Xie, Y. and Blankenship, J. T. (2018). Differentially-dimensioned furrow formation by zygotic gene expression and the MBT. PLoS Genet 14(1): e1007174. PubMed ID: 29337989
Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. This study presents an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. The deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and the X-chromosomal contribution was tracked to furrow lengthening to the nullo gene product. Checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, the turnover of maternal gene products was examined, and this was found to be a minor contributor to the developmental regulation of furrow morphologies. These results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis.
Auld, A. L., Roberts, S. A., Murphy, C. B., Camuglia, J. M. and Folker, E. S. (2018). Aplip1, the Drosophila homolog of JIP1, regulates myonuclear positioning and muscle stability. J Cell Sci 131(6). PubMed ID: 29487176
During muscle development, myonuclei undergo a complex set of movements that result in evenly spaced nuclei throughout the muscle cell. In Drosophila, two separate pools of Kinesin and Dynein work in synchrony to drive this process. However, how these two pools are specified is not known. This study investigate the role of Aplip1 (the Drosophila homolog of JIP1, JIP1 is also known as MAPK8IP1), a known regulator of both Kinesin and Dynein, in myonuclear positioning. Aplip1 localizes to the myotendinous junction and has genetically separable roles in myonuclear positioning and muscle stability. In Aplip1 mutant embryos, there was an increase in the percentage of embryos that had both missing and collapsed muscles. Via a separate mechanism, it was demonstrated that Aplip1 regulates both the final position of and the dynamic movements of myonuclei. Aplip1 genetically interacts with both Raps (also known as Pins) and Kinesin to position myonuclei. Furthermore, Dynein and Kinesin localization are disrupted in Aplip1 mutants suggesting that Aplip1-dependent nuclear positioning requires Dynein and Kinesin. Taken together, these data are consistent with Aplip1 having a function in the regulation of Dynein- and Kinesin-mediated pulling of nuclei from the muscle end.
Zmojdzian, M., de Joussineau, S., Da Ponte, J. P. and Jagla, K. (2018). Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila. Development 145(2). PubMed ID: 29247145
The Drosophila heart, composed of discrete subsets of cardioblasts and pericardial cells, undergoes Hox-triggered anterior-posterior morphogenesis, leading to a functional subdivision into heart proper and aorta, with its most anterior part forming a funnel-shaped cardiac outflow. Cardioblasts differentiate into Tin-positive 'working myocytes' and Svp-expressing ostial cells. However, developmental fates and functions of heart-associated pericardial cells remain elusive. This study shows that the pericardial cells that express the transcription factor Even Skipped adopt distinct fates along the anterior-posterior axis. Among them, the most anterior Antp-Ubx-AbdA-negative cells form a novel cardiac outflow component that is called the outflow hanging structure, whereas the Antp-expressing cells differentiate into wing heart precursors. Interestingly, Hox gene expression in the Even Skipped-positive cells not only underlies their antero-posterior diversification, but also influences heart morphogenesis in a non-cell-autonomous way. In brief, this study has identified a new cardiac outflow component derived from a subset of Even Skipped-expressing cells that stabilises the anterior heart tip, and demonstrate non-cell-autonomous effects of Hox gene expression in the Even Skipped-positive cells on heart morphogenesis.
Schmidt, A., Lv, Z. and Grosshans, J. (2018). ELMO and Sponge specify subapical restriction of Canoe and formation of the subapical domain in early Drosophila embryos. Development 145(2). PubMed ID: 29361564
Canoe/Afadin and the GTPase Rap1 specify the subapical domain during cellularization in Drosophila embryos. The timing of domain formation is unclear. The subapical domain might gradually mature or emerge synchronously with the basal and lateral domains. The potential mechanism for activation of Rap1 by guanyl nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs) is unknown. This study retraced the emergence of the subapical domain at the onset of cellularization by in vivo imaging with CanoeYFP in comparison to the lateral and basal markers ScribbledGFP and CherrySlam. CanoeYFP accumulates at a subapical position at about the same time as the lateral marker ScribbledGFP but a few minutes prior to basal CherrySlam. Furthermore, the unconventional GEF complex ELMO (Ced-12)-Sponge is subapically enriched and is required for subapical restriction of Canoe. The localization dynamics of ELMO-Sponge suggests a patterning mechanism for positioning the subapical region adjacent to the apical region. While marking the disc-like apical regions before cellularization, ELMO-Sponge redistributes to a ring-like pattern surrounding the apical region at the onset of cellularization.
Ansari, S., Troelenberg, N., Dao, V. A., Richter, T., Bucher, G. and Klingler, M. (2018). Double abdomen in a short-germ insect: Zygotic control of axis formation revealed in the beetle Tribolium castaneum. Proc Natl Acad Sci U S A 115(8): 1819-1824. PubMed ID: 29432152
Evolutionary Homolog Study
The distinction of anterior versus posterior is a crucial first step in animal embryogenesis. In Drosophila, this axis is established by morphogenetic gradients contributed by the mother that regulate zygotic target genes. This principle has been considered to hold true for insects in general. This study investigated symmetry breaking in the beetle Tribolium castaneum, which among insects represents the more ancestral short-germ embryogenesis. Maternal Tc-germ cell-less is required for anterior localization of maternal Tc-axin, which represses Wnt signaling and promotes expression of anterior zygotic genes. Both RNAi targeting Tc-germ cell-less or double RNAi knocking down the zygotic genes Tc-homeobrain and Tc-zen1 led to the formation of a second growth zone at the anterior, which resulted in double-abdomen phenotypes. Conversely, interfering with two posterior factors, Tc-caudal and Wnt, caused double-anterior phenotypes. These findings reveal that maternal and zygotic mechanisms, including Wnt signaling, are required for establishing embryo polarity and induce the segmentation clock in a short-germ insect.
Rosa, J. B., Metzstein, M. M. and Ghabrial, A. S. (2018). An Ichor-dependent apical extracellular matrix regulates seamless tube shape and integrity. PLoS Genet 14(1): e1007146. PubMed ID: 29309404
During sprouting angiogenesis in the vertebrate vascular system, and primary branching in the Drosophila tracheal system, specialized tip cells direct branch outgrowth and network formation. When tip cells lumenize, they form subcellular (seamless) tubes. How these seamless tubes are made, shaped and maintained remains poorly understood. This study characterize a Drosophila mutant called ichor (ich), and show that ich is essential for the integrity and shape of seamless tubes in tracheal terminal cells. Ich regulates seamless tubulogenesis via its role in promoting the formation of a mature apical extracellular matrix (aECM) lining the lumen of the seamless tubes. ich encodes a zinc finger protein (CG11966) that acts, as a transcriptional activator required for the expression of multiple aECM factors, including a novel membrane-anchored trypsin protease (CG8213). Thus, the integrity and shape of seamless tubes are regulated by the aECM that lines their lumens.

Monday, March 26th

Brady, M. M., McMahan, S. and Sekelsky, J. (2018). Loss of Drosophila Mei-41/ATR alters meiotic crossover patterning. Genetics 208(2): 579-588. PubMed ID: 29247012
Meiotic crossovers must be properly patterned to ensure accurate disjunction of homologous chromosomes during meiosis I. Disruption of the spatial distribution of crossovers can lead to nondisjunction, aneuploidy, gamete dysfunction, miscarriage, or birth defects. One of the earliest identified genes involved in proper crossover patterning is Drosophila mei-41, which encodes the ortholog of the checkpoint kinase ATR. Analysis of hypomorphic mutants suggested the existence of crossover patterning defects, but it was not possible to assess this in null mutants because of maternal-effect embryonic lethality. To overcome this lethality, mei-41 null mutants were constructed in which wild-type Mei-41 was expressed in the germline after completion of meiotic recombination, allowing progeny to survive. Crossovers were decreased to about one-third of wild-type levels, but the reduction is not uniform, being less severe in the proximal regions of chromosome 2L than in medial or distal 2L or on the X chromosome. None of the crossovers formed in the absence of Mei-41 require Mei-9, the presumptive meiotic resolvase, suggesting that Mei-41 functions everywhere, despite the differential effects on crossover frequency. Interference appears to be significantly reduced or absent in mei-41 mutants, but the reduction in crossover density in centromere-proximal regions is largely intact. It is proposed that crossover patterning is achieved in a stepwise manner, with the crossover suppression related to proximity to the centromere occurring prior to and independently of crossover designation and enforcement of interference. In this model, Mei-41 has an essential function in meiotic recombination after the centromere effect is established but before crossover designation and interference occur.
Kim, M., Ekhteraei-Tousi, S., Lewerentz, J. and Larsson, J. (2018). The X-linked 1.688 satellite in Drosophila melanogaster promotes specific targeting by painting of fourth. Genetics 208(2): 623-632. PubMed ID: 29242291
Repetitive DNA, represented by transposons and satellite DNA, constitutes a large portion of eukaryotic genomes, being the major component of constitutive heterochromatin. There is a growing body of evidence that it regulates several nuclear functions including chromatin state and the proper functioning of centromeres and telomeres. The 1.688 satellite is one of the most abundant repetitive sequences in Drosophila melanogaster, with the longest array being located in the pericentromeric region of the X-chromosome. Short arrays of 1.688 repeats are widespread within the euchromatic part of the X-chromosome, and these arrays were recently suggested to assist in recognition of the X-chromosome by the dosage compensation male-specific lethal complex. A short array of 1.688 satellite repeats is essential for recruitment of the protein POF to a previously described site on the X-chromosome (PoX2) and to various transgenic constructs. On an isolated target, i.e., an autosomic transgene consisting of a gene upstream of 1.688 satellite repeats, POF is recruited to the transgene in both males and females. The sequence of the satellite, as well as its length and position within the recruitment element, are the major determinants of targeting. Moreover, the 1.688 array promotes POF targeting to the roX1-proximal PoX1 site in trans. Finally, binding of POF to the 1.688-related satellite-enriched sequences is conserved in evolution. It is hypothesized that the 1.688 satellite functioned in an ancient dosage compensation system involving POF targeting to the X-chromosome.
Wang, Q., Sun, Q., Czajkowsky, D. M. and Shao, Z. (2018). Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells. Nat Commun 9(1): 188. PubMed ID: 29335463
Topologically associating domains (TADs) are fundamental elements of the eukaryotic genomic structure. However, recent studies suggest that the insulating complexes, CTCF/cohesin, present at TAD borders in mammals are absent from those in Drosophila melanogaster, raising the possibility that border elements are not conserved among metazoans. Using in situ Hi-C with sub-kb resolution, this study shows that the D. melanogaster genome is almost completely partitioned into >4000 TADs, nearly sevenfold more than previously identified. The overwhelming majority of these TADs are demarcated by the insulator complexes, BEAF-32/CP190, or BEAF-32/Chromator, indicating that these proteins may play an analogous role in flies as that of CTCF/cohesin in mammals. Moreover, extended regions previously thought to be unstructured are shown to consist of small contiguous TADs, a property also observed in mammals upon re-examination. Altogether, this work demonstrates that fundamental features associated with the higher-order folding of the genome are conserved from insects to mammals.
Aughey, G. N., Estacio Gomez, A., Thomson, J., Yin, H. and Southall, T. D. (2018). CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo. Elife 7. PubMed ID: 29481322
During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. This study shows that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). Chromatin accessibility was profiled in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type-specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, global changes were shown in the accessibility of chromatin between stem-cells and their differentiated progeny. These results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development.
Masuko, K., Fuse, N., Komaba, K., Katsuyama, T., Nakajima, R., Furuhashi, H. and Kurata, S. (2018). winged eye induces transdetermination of Drosophila imaginal disc by acting in concert with a histone methyltransferase, Su(var)3-9. Cell Rep 22(1): 206-217. PubMed ID: 29298422
Drosophila imaginal disc cells exhibit a remarkable ability to convert cell fates in response to various perturbations, a phenomenon called transdetermination (TD). winged eye (wge) has been identified as a factor that induces eye-to-wing TD upon overexpression in eye imaginal discs, but the molecular mechanisms underlying TD have remained largely unclear. This study found that wge induces various histone modifications and enhances the methylation of Lys9 on histone H3 (H3K9), a feature of heterochromatin. A histone methyltransferase, Su(var)3-9, is required for wge-mediated H3K9 methylation and eye-to-wing TD. Su(var)3-9 is also required for classical wound-induced TD but not for normal development, suggesting its involvement in several types of imaginal disc TDs. Transcriptome analysis revealed that wge represses eye identity genes independently of Su(var)3-9 and activates TD-related genes by acting together with Su(var)3-9. These findings provide new insights into diverse types of chromatin regulation at progressive steps of cell-fate conversions.
Ramirez, F., Bhardwaj, V., Arrigoni, L., Lam, K. C., Gruning, B. A., Villaveces, J., Habermann, B., Akhtar, A. and Manke, T. (2018). High-resolution TADs reveal DNA sequences underlying genome organization in flies. Nat Commun 9(1): 189. PubMed ID: 29335486
Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. This study usd HiCExplorer to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. Eight DNA motifs enriched at boundaries were identified, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. Boundaries can be accurately predicted using only the motif sequences at open chromatin sites. It is proposed that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, an interactive online database is presented to access and explore the spatial organization of fly, mouse and human genomes.

Friday, March 23rd

Nag, R. N., Niggli, S., Sousa-Guimaraes, S., Vazquez-Pianzola, P. and Suter, B. (2018). Mms19 is a mitotic gene that permits Cdk7 to be fully active as a Cdk-activating kinase. Development 145(2). PubMed ID: 29361561
Mms19 encodes a cytosolic iron-sulphur assembly component. This study found that Drosophila Mms19 is also essential for mitotic divisions and for the proliferation of diploid cells. Reduced Mms19 activity causes severe mitotic defects in spindle dynamics and chromosome segregation, and loss of zygotic Mms19 prevents the formation of imaginal discs. The lack of mitotic tissue in Mms19(P/P) larvae can be rescued by overexpression of the Cdk-activating kinase (CAK) complex, an activator of mitotic Cdk1, suggesting that Mms19 functions in mitosis to allow CAK (Cdk7/Cyclin H/Mat1) to become fully active as a Cdk1-activating kinase. When bound to Xpd and TFIIH, the CAK subunit Cdk7 phosphorylates transcriptional targets and not cell cycle Cdks. In contrast, free CAK phosphorylates and activates Cdk1. Physical and genetic interaction studies between Mms19 and Xpd suggest that their interaction prevents Xpd from binding to the CAK complex. Xpd bound to Mms19 therefore frees CAK complexes, allowing them to phosphorylate Cdk1 and facilitating progression to metaphase. The structural basis for the competitive interaction with Xpd seems to be the binding of Mms19, core TFIIH and CAK to neighbouring or overlapping regions of Xpd.
Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Abdelaziz, M., Debski, J., Dadlez, M. and Glover, D. M. (2017). Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation. Open Biol 7(12). PubMed ID: 29263250
The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL. Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. This study shows that in Drosophila, Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When the auto-destruction of Plk4 is overriden, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. This study shows that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region termed the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6.
Abeysundara, N., Simmonds, A. J. and Hughes, S. C. (2017). Moesin is involved in polarity maintenance and cortical remodelling during asymmetric cell division. Mol Biol Cell [Epub ahead of print]. PubMed ID: 29282284
An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts. However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. This study finds that the actin-binding protein, Moesin, is essential for neuroblast proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. These findings reveal Moesin as a novel apical polarity protein that drives cortical remodelling of dividing neuroblasts, which is essential for polarity maintenance and initial establishment of cell size asymmetry.
Guarner, A., Morris, R., Korenjak, M., Boukhali, M., Zappia, M. P., Van Rechem, C., Whetstine, J. R., Ramaswamy, S., Zou, L., Frolov, M. V., Haas, W. and Dyson, N. J. (2017). E2F/DP prevents cell-cycle progression in endocycling fat body cells by suppressing dATM expression. Dev Cell 43(6): 689-703.e685. PubMed ID: 29233476
To understand the consequences of the complete elimination of E2F regulation, the proteome was profiled of Drosophila dDP mutants that lack functional E2F/DP complexes. The results uncovered changes in the larval fat body, a differentiated tissue that grows via endocycles. An unexpected mechanism of E2F/DP action is reported that promotes quiescence in this tissue. In the fat body, dE2F/dDP limits cell-cycle progression by suppressing DNA damage responses. Loss of dDP upregulates dATM, allowing cells to sense and repair DNA damage and increasing replication of loci that are normally under-replicated in wild-type tissues. Genetic experiments show that ectopic dATM is sufficient to promote DNA synthesis in wild-type fat body cells. Strikingly, reducing dATM levels in dDP-deficient fat bodies restores cell-cycle control, improves tissue morphology, and extends animal development. These results show that, in some cellular contexts, dE2F/dDP-dependent suppression of DNA damage signaling is key for cell-cycle control and needed for normal development.
Zhang, P., Pei, C., Wang, X., Xiang, J., Sun, B. F., Cheng, Y., Qi, X., Marchetti, M., Xu, J. W., Sun, Y. P., Edgar, B. A. and Yuan, Z. (2017). A balance of Yki/Sd activator and E2F1/Sd repressor complexes controls cell survival and affects organ size. Dev Cell 43(5): 603-617.e605. PubMed ID: 29207260
The Hippo/Yki and RB/E2F pathways both regulate tissue growth by affecting cell proliferation and survival, but interactions between these parallel control systems are poorly defined. This study demonstrates that interaction between Drosophila E2F1 and Sd disrupts Yki/Sd complex formation and thereby suppresses Yki target gene expression. RBF modifies these effects by reducing E2F1/Sd interaction. This regulation has significant effects on apoptosis, organ size, and progenitor cell proliferation. Using a combination of DamID-seq and RNA-seq, this study identified a set of Yki targets that play a diversity of roles during development and are suppressed by E2F1. Further, it was found that human E2F1 competes with YAP for TEAD1 binding, affecting YAP activity, indicating that this mode of cross-regulation is conserved. In sum, this study uncovers a previously unknown mechanism in which RBF and E2F1 modify Hippo signaling responses to modulate apoptosis, organ growth, and homeostasis.
Zouaz, A., Fernando, C., Perez, Y., Sardet, C., Julien, E. and Grimaud, C. (2018). Cell-cycle regulation of non-enzymatic functions of the Drosophila methyltransferase PR-Set7. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 29373730
Tight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is also targeted by the SCFbeta-TRCP ligase, but the role of this additional regulatory pathway remains unclear. This study shows that Drosophila PR-Set7 undergoes a cell-cycle proteolytic regulation, independently of its interaction with PCNA. Instead, Slimb, the ortholog of beta-TRCP, is specifically required for the degradation of the nuclear pool of PR-Set7 prior to S phase. Consequently, inactivation of Slimb leads to nuclear accumulation of PR-Set7, which triggers aberrant chromatin compaction and G1/S arrest. Strikingly, these phenotypes result from non-enzymatic PR-Set7 functions that prevent proper histone H4 acetylation independently of H4K20 methylation. Altogether, these results identify the Slimb-mediated PR-Set7 proteolysis as a new critical regulatory mechanism required for proper interphase chromatin organization at G1/S transition.

Thursday, March 22nd

Wang, F., Minakhina, S., Tran, H., Changela, N., Kramer, J. and Steward, R. (2018). Tet protein function during Drosophila development. PLoS One 13(1): e0190367. PubMed ID: 29324752
The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. This study characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. Tet was shown to be essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, the function of Tet in ovarian morphogenesis is reported. Together, these findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.
Mikoluk, C., Nagengast, A. A. and DiAngelo, J. R. (2018). The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage. Biochem Biophys Res Commun 495(1): 1528-1533. PubMed ID: 29203241
Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. This lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. This study has uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2(dsRNA) in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2(dsRNA) flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.
Yatsenko, A. S. and Shcherbata, H. R. (2018). Stereotypical architecture of the stem cell niche is spatiotemporally established by miR-125-dependent coordination of Notch and steroid signaling. Development 145(3). PubMed ID: 29361571
Stem cell niches act as signaling platforms that regulate stem cell self-renewal and sustain stem cells throughout life; however, the specific developmental events controlling their assembly are not well understood. This study shows that during Drosophila ovarian germline stem cell niche formation, the status of Notch signaling in the cell can be reprogrammed. This is controlled via steroid-induced miR-125, which targets a negative regulator of Notch signaling, Tom. Thus, miR-125 acts as a spatiotemporal coordinator between paracrine Notch and endocrine steroid signaling. Moreover, a dual security mechanism for Notch signaling activation exists to ensure the robustness of niche assembly. Particularly, stem cell niche cells can be specified either via lateral inhibition, in which a niche cell precursor acquires Notch signal-sending status randomly, or via peripheral induction, whereby Delta is produced by a specific cell. When one mechanism is perturbed due to mutations, developmental defects, or environmental stress, the remaining mechanism ensures that the niche is formed, perhaps abnormal but still functional. This guarantees that the germline stem cells will have their residence, thereby securing progressive oogenesis, thus, organism reproduction.
Ueda, M., Sato, T., Ohkawa, Y. and Inoue, Y. H. (2018). Identification of miR-305, a microRNA that promotes aging, and its target mRNAs in Drosophila. Genes Cells [Epub ahead of print]. PubMed ID: 29314553
MicroRNAs (miRNAs) are involved in the regulation of important biological processes. This study describes a novel Drosophila miRNAs involved in aging. This study selected eight Drosophila miRNAs, displaying high homology with seed sequences of aging-related miRNAs characterized in other species, and investigated whether the over-expression of these miRNAs affected aging in Drosophila adult flies. The lifespan of adults over-expressing miR-305, a miRNA showing high homology with miR-239 in C. elegans, was significantly shorter. Conversely, a reduction in miR-305 expression led to a longer lifespan than that in control flies. miR-305 over-expression accelerated the impairment of locomotor activity and promoted the age-dependent accumulation of poly-ubiquitinated protein aggregates in the muscle, as flies aged. Thus, this study shows that the ectopic expression of miR-305 has a deleterious effect on aging in Drosophila. RNA-Seq was performed o identify the targets of miR-305. Several mRNAs encoding antimicrobial peptides and insulin-like peptides were discovered, whose expression changed in adults upon miR-305 over-expression. It was further confirmed, by qRT-PCR, that miR-305 over-expression significantly decreases the mRNA levels of four antimicrobial peptides. As these mRNAs contain multiple sequences matching the seed sequence of miR-305, it is speculated that a reduction in target mRNA levels, caused by ectopic miRNA expression, promotes aging.
Coll, O., Guitart, T., Villalba, A., Papin, C., Simonelig, M. and Gebauer, F. (2018). Dicer-2 promotes mRNA activation through cytoplasmic polyadenylation. RNA. PubMed ID: 29317541
Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs, the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. This study identified Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. r2d2 mRNA, encoding a Dicer-2 partner in RNAi, was identified as a Dicer-2 polyadenylation target. These results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.
Silva, J., Aivio, S., Knobel, P. A., Bailey, L. J., Casali, A., Vinaixa, M., Garcia-Cao, I., Coyaud, E., Jourdain, A. A., Perez-Ferreros, P., Rojas, A. M., Antolin-Fontes, A., Samino-Gene, S., Raught, B., Gonzalez-Reyes, A., Ribas de Pouplana, L., Doherty, A. J., Yanes, O. and Stracker, T. H. (2018). EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation. Nat Cell Biol 20(2): 162-174. PubMed ID: 29335528
Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. This study characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and showed that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. These results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.

Wednesday, March 21st

Pandey, A. and Jafar-Nejad, H. (2018). Cell aggregation assays to evaluate the binding of the Drosophila Notch with trans-ligands and its inhibition by cis-ligands. J Vis Exp(131). PubMed ID: 29364239
Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. This study describes a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of >6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.
Johns, A. R., Henstridge, M. A., Saligari, M. J., Moore, K. A., Whisstock, J. C., Warr, C. G. and Johnson, T. K. (2018). Genome-wide screen for new components of the Drosophila melanogaster Torso receptor tyrosine kinase pathway. G3 (Bethesda). PubMed ID: 29363515
Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how Receptor Tyrosine Kinase (RTK) signalling is controlled. However, the mechanisms that underpin the control of Tor signalling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localises Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, a genome-wide screen was conducted to identify new pathway components that operate downstream of Tsl. Using a set of molecularly-defined chromosomal deficiencies, a screen was conducted for suppressors of ligand-dependent Tor signalling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which were mapped to the causative gene, and a further 29 that were mapped to less than 15 genes. Of the identified genes, six represent previously unknown regulators of embryonic Tor signalling. These include twins, which encodes an integral subunit of the protein phosphatase 2A complex, and alpha-tubulin at 84B, a major constituent of the microtubule network, suggesting that these may play an important role in terminal patterning. Together these data comprise a valuable resource for the discovery of new Tor pathway components. Many of these may also be required for other roles of Tor in development, such as in the larval prothoracic gland where Tor signalling controls the initiation of metamorphosis.
Petrovsky, R. and Grosshans, J. (2018). Expression of lamina proteins Lamin and Kugelkern suppresses stem cell proliferation. Nucleus 9(1): 104-118. PubMed ID: 29210315
The nuclear lamina is involved in numerous cellular functions, such as gene expression, nuclear organization, nuclear stability, and cell proliferation. The mechanism underlying the involvement of lamina is often not clear, especially in physiological or developmental contexts. This study investigated the role and activity of farnesylated lamina proteins Lamin (Lam) and Kugelkern (Kuk) in proliferation control of intestinal stem cells (ISCs) in adult Drosophila flies. ISCs mutant for Lam or kuk proliferate, whereas overexpression of Lam or Kuk strongly suppressed proliferation. The anti-proliferative activity is, at least in part, due to suppression of Jak/Stat but not Delta/Notch signaling. Lam expression suppresses Jak/Stat signaling by normalization of about 50% of the Stat target genes in ISCs.
Worley, M. I., Alexander, L. A. and Hariharan, I. K. (2018). CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs. Elife 7. PubMed ID: 29372681
Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. A screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways.
Kale, A., Ji, Z., Kiparaki, M., Blanco, J., Rimesso, G., Flibotte, S. and Baker, N. E. (2018). Ribosomal protein S12e has a distinct function in cell competition. Dev Cell 44(1): 42-55.e44. PubMed ID: 29316439
Wild-type Drosophila cells can remove cells heterozygous for ribosomal protein mutations (known as "Minute" mutant cells) from genetic mosaics, a process termed cell competition. The ribosomal protein S12 was unusual because cells heterozygous for rpS12 mutations were not competed by wild-type, and a viable missense mutation in rpS12 protected Minute cells from cell competition with wild-type cells. Furthermore, cells with Minute mutations were induced to compete with one another by altering the gene dose of rpS12, eliminating cells with more rpS12 than their neighbors. Thus RpS12 has a special function in cell competition that defines the competitiveness of cells. It is proposed that cell competition between wild-type and Minute cells is initiated by a signal of ribosomal protein haploinsufficiency mediated by RpS12. Since competition between cells expressing different levels of Myc did not require RpS12, other kinds of cell competition may be initiated differently.
Kannan, R., Cox, E., Wang, L., Kuzina, I., Gu, Q. and Giniger, E. (2018). Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance. Development 145(2). PubMed ID: 29343637
Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. This study shows that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. It furthers show that some Notch protein is tyrosine phosphorylated in Drosophila, that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, a model is proposed for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons.

Tuesday, March 20th

Santoso, C. S., Meehan, T. L., Peterson, J. S., Cedano, T. M., Turlo, C. V. and McCall, K. (2018). The ABC transporter Eato promotes cell clearance in the Drosophila melanogaster ovary. G3 (Bethesda). PubMed ID: 29295819
Two evolutionarily conserved signaling pathways that act redundantly to regulate engulfment of dead cells: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Homology searches revealed a family of putative ced-7/ABCA1 homologs encoding ABC transporters in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1, mutants were analyzed for engulfment phenotypes in oogenesis, during which nurse cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. Genetic analyses indicate that one of the ABC transporter genes, which was named Eato (CG31731), is required for nurse cell clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the follicle cells. Additionally, Eato acts in the follicle cells to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the nurse cells for their clearance. Since ABCA class transporters, such as CED-7 and Eato, are involved in lipid trafficking, it is proposed that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. This work identifies Eato as the ced-7 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during nurse cell clearance in the ovary.
Garner, S. R. C., Castellanos, M. C., Baillie, K. E., Lian, T. and Allan, D. W. (2018). Drosophila female-specific Ilp7 motoneurons are generated by Fruitless-dependent cell death in males and by a double-assurance survival role for Transformer in females. Development 145(1). PubMed ID: 29229771
Female-specific Ilp7 neuropeptide-expressing motoneurons (FS-Ilp7 motoneurons) are required in Drosophila for oviduct function in egg laying. This study uncovered cellular and genetic mechanisms underlying their female-specific generation. Programmed cell death (PCD) eliminates FS-Ilp7 motoneurons in males, and that this requires male-specific splicing of the sex-determination gene fruitless (fru) into the Fru(MC) isoform. However, in females, fru alleles that only generate Fru(M) isoforms failed to kill FS-Ilp7 motoneurons. This blockade of Fru(M)-dependent PCD was not attributable to doublesex gene function but to a non-canonical role for transformer (tra), a gene encoding the RNA splicing activator that regulates female-specific splicing of fru and dsx transcripts. In both sexes, Tra was shown to prevent PCD even when the Fru(M) isoform is expressed. In addition, it was found that Fru(MC) eliminated FS-Ilp7 motoneurons in both sexes, but only when Tra was absent. Thus, Fru(MC)-dependent PCD eliminates female-specific neurons in males, and Tra plays a double-assurance function in females to establish and reinforce the decision to generate female-specific neurons (Garner, 2018).
Wang, X. and Page-McCaw, A. (2018). Wnt6 maintains anterior escort cells as an integral component of the germline stem cell niche. Development [Epub ahead of print]. PubMed ID: 29361569
Stem cells reside in a niche, a complex cellular and molecular environment. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and this study reports that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes consequent loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. These data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap-cell produced Wnt6 survival signal.

Sugimori, S., Kumata, Y. and Kobayashi, S. (2018). Maternal Nanos-dependent RNA stabilization in the primordial germ cells of Drosophila embryos. Dev Growth Differ 60(1): 63-75. PubMed ID: 29278271
Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, this study reports that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. The level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, the region was identified required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remains in this cell type during embryogenesis. However, as expected from the reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, it is proposed that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development.
Jiang, M., Gao, Z., Wang, J. and Nurminsky, D. I. (2018). Evidence for a hierarchical transcriptional circuit in Drosophila male germline involving testis-specific TAF and two gene-specific transcription factors, Mod and Acj6. . FEBS Lett 592(1): 46-59. PubMed ID: 29235675
To analyze transcription factors involved in gene regulation by testis-specific TAF (tTAF see Cannonball), tTAF-dependent promoters were mapped and analyzed in silico. Core promoters show decreased AT content, paucity of classical promoter motifs, and enrichment with translation control element CAAAATTY. Scanning of putative regulatory regions for known position frequency matrices identified 19 transcription regulators possibly contributing to tTAF-driven gene expression. Decreased male fertility associated with mutation in one of the regulators, Acj6, indicates its involvement in male reproduction. Transcriptome study of testes from male mutants for tTAF, Acj6, and previously characterized tTAF-interacting factor Modulo implies the existence of a regulatory hierarchy of tTAF, Modulo and Acj6, in which Modulo and/or Acj6 regulate one-third of tTAF-dependent genes.
Flora, P., Schowalter, S., Wong-Deyrup, S., DeGennaro, M., Nasrallah, M. A. and Rangan, P. (2018). Transient transcriptional silencing alters the cell cycle to promote germline stem cell differentiation in Drosophila. Dev Biol 434(1): 84-95. PubMed ID: 29198563
Transcriptional silencing is a conserved process used by embryonic germ cells to repress somatic fate and maintain totipotency and immortality. In Drosophila, this transcriptional silencing is mediated by polar granule component (pgc). This study shows that in the adult ovary, pgc is required for timely germline stem cell (GSC) differentiation. Pgc is expressed transiently in the immediate GSC daughter (pre-cystoblast), where it mediates a pulse of transcriptional silencing. This transcriptional silencing mediated by pgc indirectly promotes the accumulation of Cyclin B (CycB) and cell cycle progression into late-G2 phase, when the differentiation factor bag of marbles (bam) is expressed. Pgc mediated accumulation of CycB is also required for heterochromatin deposition, which protects the germ line genome against selfish DNA elements. These results suggest that transient transcriptional silencing in the pre-cystoblast 're-programs' it away from self-renewal and toward the gamete differentiation program (Flora, 2018).

Monday, March 19th

Dohn, T. E. and Cripps, R. M. (2018). Absence of the Drosophila jump muscle actin Act79B is compensated by up-regulation of Act88F. Dev Dyn [Epub ahead of print]. PubMed ID: 29318731
Actins are structural components of the cytoskeleton and muscle, and numerous actin isoforms are found in most organisms. However, many actin isoforms are expressed in distinct patterns allowing each actin to have a specialized function. Numerous studies have demonstrated that actin isoforms both can and cannot compensate for each other under specific circumstances. This allows for an ambiguity of whether isoforms are functionally distinct. This study analyzed mutants of Drosophila Act79B, the predominant actin expressed in the adult jump muscle. Functional and structural analysis of the Act79B mutants found the flies to have normal jumping ability and sarcomere structure. Analysis of actin gene expression determined that expression of Act88F, an actin gene normally expressed in the flight muscles, was significantly up-regulated in the jump muscles of mutants. This indicated that loss of Act79B caused expansion of Act88F expression. When double mutants were created of Act79B and Act88F, this abolished the jump ability of the flies and resulted in severe defects in myofibril formation. These results indicate that Act88F can functionally substitute for Act79B in the jump muscle, and that the functional compensation in actin expression in the jump muscles only occurs through Act88F.
Tsoumpekos, G., Nemetschke, L. and Knust, E. (2018). Drosophila Big bang regulates the apical cytocortex and wing growth through junctional tension. J Cell Biol [Epub ahead of print]. PubMed ID: 29326288
Growth of epithelial tissues is regulated by a plethora of components, including signaling and scaffolding proteins, but also by junctional tension, mediated by the actomyosin cytoskeleton. However, how these players are spatially organized and functionally coordinated is not well understood. This study identify the Drosophila melanogaster scaffolding protein Big bang as a novel regulator of growth in epithelial cells of the wing disc by ensuring proper junctional tension. Loss of big bang results in the reduction of the regulatory light chain of nonmuscle myosin, Spaghetti squash. This is associated with an increased apical cell surface, decreased junctional tension, and smaller wings. Strikingly, these phenotypic traits of big bang mutant discs can be rescued by expressing constitutively active Spaghetti squash. Big bang colocalizes with Spaghetti squash in the apical cytocortex and is found in the same protein complex. These results suggest that in epithelial cells of developing wings, the scaffolding protein Big bang controls apical cytocortex organization, which is important for regulating cell shape and tissue growth.
Takeda, M., Sami, M. M. and Wang, Y. C. (2018). A homeostatic apical microtubule network shortens cells for epithelial folding via a basal polarity shift. Nat Cell Biol 20(1): 36-45. PubMed ID: 29203884
Epithelial folding is typically driven by localized actomyosin contractility. However, it remains unclear how epithelia deform when myosin levels are low and uniform. In the Drosophila gastrula, dorsal fold formation occurs despite a lack of localized myosin changes, while the fold-initiating cells reduce cell height following basal shifts of polarity via an unknown mechanism. This study shows that cell shortening depends on an apical microtubule network organized by the CAMSAP protein Patronin. Prior to gastrulation, microtubule forces generated by the minus-end motor dynein scaffold the apical cell cortex into a dome-like shape, while the severing enzyme Katanin facilitates network remodelling to ensure tissue-wide cell size homeostasis. During fold initiation, Patronin redistributes following basal polarity shifts in the initiating cells, apparently weakening the scaffolding forces to allow dome descent. The homeostatic network that ensures size/shape homogeneity is thus repurposed for cell shortening, linking epithelial polarity to folding via a microtubule-based mechanical mechanism.
Tempesta, C., Hijazi, A., Moussian, B. and Roch, F. (2017). Boudin trafficking reveals the dynamic internalisation of specific septate junction components in Drosophila. PLoS One 12(10): e0185897. PubMed ID: 28977027
The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions. Multiple studies in Drosophila have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. This study used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. This protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. The data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.
Adikes, R. C., Hallett, R. A., Saway, B. F., Kuhlman, B. and Slep, K. C. (2017). Control of microtubule dynamics using an optogenetic microtubule plus end-F-actin cross-linker. J Cell Biol [Epub ahead of print]. PubMed ID: 29259096
A novel optogenetic tool, SxIP-improved light-inducible dimer (iLID), has been developed to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein-dependent manner using blue light. SxIP-iLID was used to track MT plus ends and recruit tgRFP-SspB upon blue light activation. This study was used to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.
Lv, Z., Rosenbaum, J., Aspelmeier, T. and Grosshans, J. (2018). A 'molecular guillotine' reveals an interphase function of Kinesin-5. J Cell Sci. PubMed ID: 29361546
Motor proteins are important for transport and force generation in a variety of cellular processes and morphogenesis. This study designed a general strategy for conditional motor mutants by inserting a protease cleavage site into the 'neck' between the head domain and the stalk of the motor protein, making the protein susceptible to proteolytic cleavage at the neck by the corresponding protease. To demonstrate the feasibility of this approach, the cleavage site of TEV protease was inserted into the neck of the tetrameric motor Kinesin-5. Application of TEV protease led to a specific depletion and functional loss of Kinesin-5 in Drosophila embryos. With this approach, it was revealed that Kinesin-5 stabilized the microtubule network during interphase in syncytial embryos. The "molecular guillotine' can potentially be applied to many motor proteins due to the conserved structures of kinesins and myosins with accessible necks.

Friday, March 16th

Yoon, S., Cho, B., Shin, M., Koranteng, F., Cha, N. and Shim, J. (2017). Iron homeostasis controls myeloid blood cell differentiation in Drosophila. Mol Cells 40(12): 976-985. PubMed ID: 29237257
Iron is an essential divalent ion for aerobic life. Life has evolved to maintain iron homeostasis for normal cellular and physiological functions and therefore imbalances in iron levels exert a wide range of consequences. Responses to iron dysregulation in blood development, however, remain elusive. This study found that iron homeostasis is critical for differentiation of Drosophila blood cells in the larval hematopoietic organ, called the lymph gland. Supplementation of an iron chelator, bathophenanthroline disulfate (BPS) results in an excessive differentiation of the crystal cell in the lymph gland. This phenotype is recapitulated by loss of Fer1HCH in the intestine, indicating that reduced levels of systemic iron enhances crystal cell differentiation. Detailed analysis of Fer1HCH-tagged-GFP revealed that Fer1HCH is also expressed in the hematopoietic systems. Lastly, blocking Fer1HCH expression in the mature blood cells showed marked increase in the blood differentiation of both crystal cells and plasmatocytes. Thus, this work suggests a relevance of systemic and local iron homeostasis in blood differentiation, prompting further investigation of molecular mechanisms underlying iron regulation and cell fate determination in the hematopoietic system.
Zuber, R., Norum, M., Wang, Y., Oehl, K., Gehring, N., Accardi, D., Bartozsewski, S., Berger, J., Flotenmeyer, M. and Moussian, B. (2017). The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila. Eur J Cell Biol. PubMed ID: 29306642
Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, this study found that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu (CG9990) and the extracellular protein Snsl (CG2837). In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. These data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.
Delgado, M. G., Oliva, C., Lopez, E., Ibacache, A., Galaz, A., Delgado, R., Barros, L. F. and Sierralta, J. (2018). Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress. Sci Rep 8(1): 1186. PubMed ID: 29352169
The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. This study shows that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. These findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.
Lee, Y., Poudel, S., Kim, Y., Thakur, D. and Montell, C. (2018). Calcium taste avoidance in Drosophila. Neuron 97(1): 67-74.e64. PubMed ID: 29276056
Many animals, ranging from vinegar flies to humans, discriminate a wide range of tastants, including sugars, bitter compounds, NaCl, and sour. However, the taste of Ca(2+) is poorly understood, and it is unclear whether animals such as Drosophila melanogaster are endowed with this sense. This study examined Ca(2+) taste in Drosophila and showed that high levels of Ca(2+) are aversive. The repulsion was mediated by two mechanisms-activation of a specific class of gustatory receptor neurons (GRNs), which suppresses feeding and inhibition of sugar-activated GRNs, which normally stimulates feeding. The distaste for Ca(2+), and Ca(2+)-activated action potentials required several members of the variant ionotropic receptor (IR) family (IR25a, IR62a, and IR76b). Consistent with the Ca(2+) rejection, it was found that high concentrations of Ca(2+) decreased survival. It is concluded that gustatory detection of Ca(2+) represents an additional sense of taste in Drosophila and is required for avoiding toxic levels of this mineral.
Wood, J. G., Schwer, B., Wickremesinghe, P. C., Hartnett, D. A., Burhenn, L., Garcia, M., Li, M., Verdin, E. and Helfand, S. L. (2018). Sirt4 is a mitochondrial regulator of metabolism and lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A. PubMed ID: 29378963
Sirtuins are an evolutionarily conserved family of NAD(+)-dependent deacylases that control metabolism, stress response, genomic stability, and longevity. This study shows that the sole mitochondrial sirtuin in Drosophila melanogaster, Sirt4, regulates energy homeostasis and longevity. Sirt4 knockout flies have a short lifespan, with increased sensitivity to starvation and decreased fertility and activity. In contrast, flies overexpressing Sirt4 either ubiquitously or specifically in the fat body are long-lived. Despite rapid starvation, Sirt4 knockout flies paradoxically maintain elevated levels of energy reserves, including lipids, glycogen, and trehalose, while fasting, suggesting an inability to properly catabolize stored energy. Metabolomic analysis indicates several specific pathways are affected in Sirt4 knockout flies, including glycolysis, branched-chain amino acid metabolism, and impaired catabolism of fatty acids with chain length C18 or greater. Together, these phenotypes point to a role for Sirt4 in mediating the organismal response to fasting, and ensuring metabolic homeostasis and longevity.
Steck, K., Walker, S. J., Itskov, P. M., Baltazar, C., Moreira, J. M. and Ribeiro, C. (2018). Internal amino acid state modulates yeast taste neurons to support protein homeostasis in Drosophila. Elife 7. PubMed ID: 29393045
To optimize fitness, animals must dynamically match food choices to their current needs. For drosophilids, yeast fulfils most dietary protein and micronutrient requirements. While several yeast metabolites activate known gustatory receptor neurons (GRNs) in Drosophila melanogaster, the chemosensory channels mediating yeast feeding remain unknown. This study identified a class of proboscis GRNs required for yeast intake. Within this class, taste peg GRNs are specifically required to sustain yeast feeding. Sensillar GRNs, however, mediate feeding initiation. Furthermore, the response of yeast GRNs, but not sweet GRNs, is enhanced following deprivation from amino acids, providing a potential basis for protein-specific appetite. Although nutritional and reproductive states synergistically increase yeast appetite, reproductive state acts independently of nutritional state, modulating processing downstream of GRNs. Together, these results suggest that different internal states act at distinct levels of a dedicated gustatory circuit to elicit nutrient-specific appetites towards a complex, ecologically relevant protein source.

Thursday, March 15th

Dillard, C., Narbonne-Reveau, K., Foppolo, S., Lanet, E. and Maurange, C. (2018). Two distinct mechanisms silence chinmo in Drosophila neuroblasts and neuroepithelial cells to limit their self-renewal. Development 145(2). PubMed ID: 29361557
Whether common principles regulate the self-renewing potential of neural stem cells (NSCs) throughout the developing central nervous system is still unclear. In the Drosophila ventral nerve cord and central brain, asymmetrically dividing NSCs, called neuroblasts (NBs), progress through a series of sequentially expressed transcription factors that limits self-renewal by silencing a genetic module involving the transcription factor Chinmo. This study finds that Chinmo also promotes neuroepithelium growth in the optic lobe during early larval stages by boosting symmetric self-renewing divisions while preventing differentiation. Neuroepithelium differentiation in late larvae requires the transcriptional silencing of chinmo by ecdysone, the main steroid hormone, therefore allowing coordination of neural stem cell self-renewal with organismal growth. In contrast, chinmo silencing in NBs is post-transcriptional and does not require ecdysone. Thus, during Drosophila development, humoral cues or tissue-intrinsic temporal specification programs respectively limit self-renewal in different types of neural progenitors through the transcriptional and post-transcriptional regulation of the same transcription factor.
Upadhyay, M., Kuna, M., Tudor, S., Martino Cortez, Y. and Rangan, P. (2018). A switch in the mode of Wnt signaling orchestrates the formation of germline stem cell differentiation niche in Drosophila. PLoS Genet 14(1): e1007154. PubMed ID: 29370168
Germline stem cell (GSC) self-renewal and differentiation into gametes is regulated by both intrinsic factors in the germ line as well as extrinsic factors from the surrounding somatic niche. dWnt4, in the escort cells of the adult somatic niche promotes GSC differentiation using the canonical beta-catenin-dependent transcriptional pathway to regulate escort cell survival, adhesion to the germ line and downregulation of self-renewal signaling. This study shows that in addition to the beta-catenin-dependent canonical pathway, dWnt4 also uses downstream components of the Wnt non-canonical pathway to promote escort cell function earlier in development. The downstream non-canonical components, RhoA, Rac1 and cdc42, are expressed at high levels and are active in escort cell precursors of the female larval gonad compared to the adult somatic niche. Consistent with this expression pattern, the non-canonical pathway components were found to function in the larval stages but not in adults to regulate GSC differentiation. In the larval gonad, dWnt4, RhoA, Rac1 and cdc42 are required to promote intermingling of escort cell precursors, a function that then promotes proper escort cell function in the adults. dWnt4 acts by modulating the activity of RhoA, Rac1 and cdc42, but not their protein levels. Together, these results indicate that at different points of development, dWnt4 switches from using the non-canonical pathway components to using a beta-catenin-dependent canonical pathway in the escort cells to facilitate the proper differentiation of GSCs.
Chen, J., Xu, N., Wang, C., Huang, P., Huang, H., Jin, Z., Yu, Z., Cai, T., Jiao, R. and Xi, R. (2018). Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells. Nat Cell Biol 20(2): 152-161. PubMed ID: 29335529
The process through which multiple types of cell-lineage-restricted progenitor cells are specified from multipotent stem cells is unclear. This study shows that, in intestinal stem cell lineages in adult Drosophila, in which the Delta-Notch-signalling-guided progenitor cell differentiation into enterocytes is the default mode, the specification of enteroendocrine cells (EEs) is initiated by transient Scute activation in a process driven by transcriptional self-stimulation combined with a negative feedback regulation between Scute and Notch targets. Scute activation induces asymmetric intestinal stem cell divisions that generate EE progenitor cells. The mitosis-inducing and fate-inducing activities of Scute guide each EE progenitor cell to divide exactly once prior to its terminal differentiation, yielding a pair of EEs. The transient expression of a fate inducer therefore specifies both type and numbers of committed progenitor cells originating from stem cells, which could represent a general mechanism used for diversifying committed progenitor cells from multipotent stem cells.
Kaur, P., Saunders, T. E. and Tolwinski, N. S. (2017). Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis. Sci Rep 7(1): 16636. PubMed ID: 29192250
Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, this study investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila beta-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of beta-catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. It is anticipated that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.
Haller, S., Kapuria, S., Riley, R. R., O'Leary, M. N., Schreiber, K. H., Andersen, J. K., Melov, S., Que, J., Rando, T. A., Rock, J., Kennedy, B. K., Rodgers, J. T. and Jasper, H. (2017). mTORC1 activation during repeated regeneration impairs somatic stem cell maintenance. Cell Stem Cell 21(6): 806-818.e805. PubMed ID: 29220665
The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. This study shows that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.
Speder, P. and Brand, A. H. (2018). Systemic and local cues drive neural stem cell niche remodelling during neurogenesis in Drosophila. Elife 7. PubMed ID: 29299997
Successful neurogenesis requires adequate proliferation of neural stem cells (NSCs) and their progeny, followed by neuronal differentiation, maturation and survival. NSCs inhabit a complex cellular microenvironment, the niche, which influences their behaviour. To ensure sustained neurogenesis, niche cells must respond to extrinsic, environmental changes whilst fulfilling the intrinsic requirements of the neurogenic program and adapting their roles accordingly. However, very little is known about how different niche cells adjust their properties to such inputs. This study shows that nutritional and NSC-derived signals induce the remodelling of Drosophila cortex glia, adapting this glial niche to the evolving needs of NSCs. First, nutrition-induced activation of PI3K/Akt drives the cortex glia to expand their membrane processes. Second, when NSCs emerge from quiescence to resume proliferation, they signal to glia to promote membrane remodelling and the formation of a bespoke structure around each NSC lineage. The remodelled glial niche is essential for newborn neuron survival.

Wednesday, March 14

Wei, K. H., Lower, S. E., Caldas, I. V., Sless, T. J., Barbash, D. A. and Clark, A. G. (2018). Variable rates of simple satellite gains across the Drosophila phylogeny. Mol Biol Evol [Epub ahead of print]. PubMed ID: 29361128
Simple satellites are tandemly repeating short DNA motifs that can span megabases in eukaryotic genomes. Because they can cause genomic instability through non-allelic homologous exchange, they are primarily found in the repressive heterochromatin near centromeres and telomeres where recombination is minimal, and on the Y chromosome, where they accumulate as the chromosome degenerates. Interestingly, the types and abundances of simple satellites often vary dramatically between closely related species, suggesting that they turn over rapidly. However, limited sampling has prevented detailed understanding of their evolutionary dynamics. This study characterized simple satellites from whole-genome sequences generated from males and females of nine Drosophila species, spanning 40 million years of evolution. PCR-free library preparation and post-sequencing GC-correction better capture satellite quantities than conventional methods. Over half of the 207 simple satellites identified are species-specific, consistent with previous descriptions of their rapid evolution. Based on a maximum parsimony framework, it was determined that most interspecific differences are due to lineage-specific gains. Simple satellites gained within a species are typically a single mutation away from abundant existing satellites, suggesting that they likely emerge from existing satellites, especially in the genomes of satellite-rich species. Interestingly, unlike most of the other lineages which experience various degrees of gains, the lineage leading up to the satellite-poor D. pseudoobscura and D. persimilis appears to be recalcitrant to gains, providing a counterpoint to the notion that simple satellites are universally rapidly evolving.
Campos, J. L., Johnston, K. and Charlesworth, B. (2017). The effects of sex-biased gene expression and X-linkage on rates of sequence evolution in Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 29228267
A faster rate of adaptive evolution of X-linked genes compared with autosomal genes (the faster-X effect) can be caused by the fixation of recessive or partially recessive advantageous mutations. This effect should be largest for advantageous mutations that affect only male fitness, and least for mutations that affect only female fitness. These predictions were tested in Drosophila melanogaster by using coding and functionally significant non-coding sequences of genes with different levels of sex-biased expression. Consistent with theory, nonsynonymous substitutions in most male-biased and unbiased genes show faster adaptive evolution on the X. However, genes with very low recombination rates do not show such an effect, possibly as a consequence of Hill-Robertson interference. Contrary to expectation, there was a substantial faster-X effect for female-biased genes. After correcting for recombination rate differences, however, female-biased genes did not show a faster X-effect. Similar analyses of non-coding UTRs and long introns showed a faster-X effect for all groups of genes, other than introns of female-biased genes. Given the strong evidence that deleterious mutations are mostly recessive or partially recessive, a slower rate of evolution of X-linked genes would be expected for slightly deleterious mutations that become fixed by genetic drift. Surprisingly, little evidence for this was found after correcting for recombination rate, implying that weakly deleterious mutations are mostly close to being semidominant. This is consistent with evidence from polymorphism data, which is used to test how models of selection that assume semidominance with no sex-specific fitness effects may bias estimates of purifying selection.
Wensing, K. U., Koppik, M. and Fricke, C. (2017). Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster. Ecol Evol 7(23): 10361-10378. PubMed ID: 29238561
Competition between males creates potential for pre- and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. This study evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. In a setting where males competed directly with a rival male for access to a female and fertilization of her ova, polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, no difference was found in male sperm defense competitiveness between the different selection regimes. Instead, monogamous males were found to be inferior in precopulatory competition, which indicates that in this system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. These findings are discussed in the context of findings from previous experimental evolution studies in Drosophila and other invertebrate species.
Noreen, S., Pegoraro, M., Nouroz, F., Tauber, E. and Kyriacou, C. P. (2018). Interspecific studies of circadian genes period and timeless in Drosophila. Gene [Epub ahead of print]. PubMed ID: 29353056
The level of rescue of clock function in genetically arrhythmic Drosophila melanogaster hosts using interspecific clock gene transformation was used to study the putative intermolecular coevolution between interacting clock proteins. Among them PER and TIM are the two important negative regulators of the circadian clock feedback loop. This study transformed either the D. pseudoobscura per or tim transgenes into the corresponding arrhythmic D. melanogaster mutant (per01 or tim01) and observed >50% rhythmicity but the period of activity rhythm was either longer (D. pseudoobscura-per) or shorter than 24h (D. pseudoobscura-tim) compared to controls. By introducing both transgenes simultaneously into double mutants, it was observed that the period of the activity rhythm was rescued by the pair of hemizygous transgenes (~24h). These flies also showed a more optimal level of temperature compensation for the period. Under LD 12:12 these flies have a D. pseudoobscura like activity profile with the absence of morning anticipation as well as a very prominent earlier evening peak of activity rhythm. These observation are consistent with the view that TIM and PER form a heterospecific coevolved module at least for the circadian period of activity rhythms. However the strength of rhythmicity was reduced by having both transgenes present, so while evidence for a coevolution between PER and TIM is observed for some characters it is not for others.
Talbert, P., Kasinathan, S. and Henikoff, S. (2018). Simple and complex centromeric satellites in Drosophila sibling species. Genetics [Epub ahead of print]. PubMed ID: 29305387
Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats, often 100-400 bp in length, found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5-12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. This study use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5-bp and 10-bp repeats, but in closely related D. simulans, more complex repeats comprise the centromeres. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster.
Rice, G., Barmina, O., Hu, K. and Kopp, A. (2018). Evolving doublesex expression correlates with the origin and diversification of male sexual ornaments in the Drosophila immigrans species group. Evol Dev 20(2):78-88. PubMed ID: 29372584
Male ornaments and other sex-specific traits present some of the most dramatic examples of evolutionary innovations. Comparative studies of similar but independently evolved traits are particularly important for identifying repeated patterns in the evolution of these traits. Male-specific modifications of the front legs have evolved repeatedly in Drosophilidae and other Diptera. The best understood of these novel structures is the sex comb of Drosophila melanogaster and its close relatives. This study examined the evolution of another male foreleg modification, the sex brush, found in the distantly related Drosophila immigrans species group. Similar to the sex comb, it was found that the origin of the sex brush correlates with novel, spatially restricted expression of the doublesex (dsx) transcription factor, the primary effector of the Drosophila sex determination pathway. The diversity of Dsx expression patterns in the immigrans species group closely reflects the differences in the presence, position, and size of the sex brush. Together with previous work on sex comb evolution, these observations suggest that tissue-specific activation of dsx expression may be a common mechanism responsible for the evolution of sexual dimorphism and particularly for the origin of novel male-specific ornaments.
Veltsos, P., Fang, Y., Cossins, A. R., Snook, R. R. and Ritchie, M. G. (2017). Mating system manipulation and the evolution of sex-biased gene expression in Drosophila. Nat Commun 8(1): 2072. PubMed ID: 29233985
Sex differences in dioecious animals are pervasive and result from gene expression differences. Elevated sexual selection has been predicted to increase the number and expression of male-biased genes, and experimentally imposing monogamy on Drosophila melanogaster has led to a relative feminisation of the transcriptome. This hypothesis was further tested by subjecting another polyandrous species, D. pseudoobscura, to 150 generations of experimental monogamy or elevated polyandry. Sex-biased genes were found to change in expression but, contrary to predictions, there is usually masculinisation of the transcriptome under monogamy, although this depends on tissue and sex. Gene expression changes were identified and described following courtship experience. Courtship often influences gene expression, including patterns in sex-biased gene expression. These results confirm that mating system manipulation disproportionately influences sex-biased gene expression but show that the direction of change is dynamic and unpredictable.
Jayaswal, V., Jimenez, J., Magie, R., Nguyen, K., Clifton, B., Yeh, S. and Ranz, J. M. (2018). A species-specific multigene family mediates differential sperm displacement in Drosophila melanogaster. Evolution 72(2): 399-403. PubMed ID: 29315521
Sperm competition is a postcopulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. This study compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. A less effective removal of first-to-mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. These results highlight the role young genes can play in driving the evolution of sperm competition.

Tuesday, March 13th

Salis, P., Payre, F., Valenti, P., Bazellieres, E., Le Bivic, A. and Mottola, G. (2017). Crumbs, Moesin and Yurt regulate junctional stability and dynamics for a proper morphogenesis of the Drosophila pupal wing epithelium. Sci Rep 7(1): 16778. PubMed ID: 29196707
The Crumbs (Crb) complex is a key epithelial determinant. To understand its role in morphogenesis, this study examined its function in the Drosophila pupal wing, an epithelium undergoing hexagonal packing and formation of planar-oriented hairs. Crb distribution is dynamic, being stabilized to the subapical region just before hair formation. Lack of crb or stardust, but not DPatj, affects hexagonal packing and delays hair formation, without impairing epithelial polarities but with increased fluctuations in cell junctions and perimeter length, fragmentation of adherens junctions and the actomyosin cytoskeleton. Crb interacts with Moesin and Yurt, FERM proteins regulating the actomyosin network. Moesin and Yurt distribution at the subapical region depends on Crb. In contrast to previous reports, yurt, but not moesin, mutants phenocopy crb junctional defects. Moreover, while unaffected in crb mutants, cell perimeter increases in yurt mutant cells and decreases in the absence of moesin function. These data suggest that Crb coordinates proper hexagonal packing and hair formation, by modulating junction integrity via Yurt and stabilizing cell perimeter via both Yurt and Moesin. The Drosophila pupal wing thus appears as a useful system to investigate the functional diversification of the Crb complex during morphogenesis, independently of its role in polarity.
Conway, S., Sansone, C. L., Benske, A., Kentala, K., Billen, J., Broeck, J. V. and Blumenthal, E. M. (2018). Pleiotropic and novel phenotypes in the Drosophila gut caused by mutation of drop-dead. J Insect Physiol. PubMed ID: 29371099
Normal gut function is vital for animal survival. In Drosophila, mutation of the gene drop-dead (drd) results in defective gut function, as measured by enlargement of the crop and reduced food movement through the gut, and drd mutation also causes the unrelated phenotypes of neurodegeneration, early adult lethality and female sterility. This work shows that adult drd mutant flies lack the peritrophic matrix (PM), an extracellular barrier that lines the lumen of the midgut and is found in many insects including flies, mosquitos and termites. The use of a drd-gal4 construct to drive a GFP reporter in late pupae and adults revealed drd expression in the anterior cardia, which is the site of PM synthesis in Drosophila. Moreover, the ability of drd knockdown or rescue with several gal4 drivers to recapitulate or rescue the gut phenotypes (lack of a PM, reduced defecation, and reduced adult survival 10-40 days post-eclosion) was correlated to the level of expression of each driver in the anterior cardia. Surprisingly, however, knocking down drd expression only in adult flies, which has previously been shown not to affect survival, eliminated the PM without reducing defecation rate. These results demonstrate that drd mutant flies have a novel phenotype, the absence of a PM, which is functionally separable from the previously described gut dysfunction observed in these flies. As the first mutant Drosophila strain reported to lack a PM, drd mutants will be a useful tool for studying the synthesis of this structure.
Ashe, S., Malhotra, V. and Raghu, P. (2018). Protein kinase D regulates metabolism and growth by controlling secretion of insulin like peptide. Dev Biol 434(1): 175-185. PubMed ID: 29247620
Mechanisms coupling growth and metabolism are conserved in Drosophila and mammals. In metazoans, such coupling is achieved across tissue scales through the regulated secretion of chemical messengers such as insulin that control the metabolism and growth of cells. Although the regulated secretion of Insulin like peptide (dILP) is key to normal growth and metabolism in Drosophila, the sub-cellular mechanisms that regulate dILP release remain poorly understood. This study found that reduced function of the only protein kinase D in Drosophila (dPKD(H)) results in delayed larval growth and development associated with abnormal sugar and lipid metabolism, reduced insulin signalling and accumulation of dILP2 in the neurosecretory IPCs of the larval brain. These phenotypes are rescued by tissue-selective reconstitution of dPKD in the neurosecretory cells of dPKD(H). Selective downregulation of dPKD activity in the neurosecretory IPCs phenocopies the growth defects, metabolic abnormalities and dILP2 accumulation seen in dPKD(H). Thus, dPKD mediated secretion of dILP2 from neurosecretory cells during development is necessary for normal larval growth.
Liu, S., Li, K., Gao, Y., Liu, X., Chen, W., Ge, W., Feng, Q., Palli, S. R. and Li, S. (2018). Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila. Proc Natl Acad Sci U S A 115(1): 139-144. PubMed ID: 29255055
In insects, the steroid hormone, 20-hydroxyecdysone (20E), elicits metamorphosis, thus promoting this transition, while the sesquiterpenoid juvenile hormone (JH) antagonizes 20E signaling to prevent precocious metamorphosis during the larval stages. However, not much is known about the mechanisms involved in cross-talk between these two hormones. This study discovered that in the ring gland (RG) of Drosophila larvae, JH and 20E control each other's biosynthesis. JH induces expression of a Kruppel-like transcription factor gene Kr-h1 in the prothoracic gland (PG), a portion of the RG that produces the 20E precursor ecdysone. By reducing both steroidogenesis autoregulation and PG size, high levels of Kr-h1 in the PG inhibit ecdysteriod biosynthesis, thus maintaining juvenile status. JH biosynthesis is prevented by 20E in the corpus allatum, the other portion of the RG that produces JH, to ensure the occurrence of metamorphosis. Hence, antagonistic actions of JH and 20E within the RG determine developmental transitions in Drosophila. This study proposes a mechanism of cross-talk between the two major hormones in the regulation of insect metamorphosis.
Ketosugbo, K. F., Bushnell, H. L. and Johnson, R. I. (2017). A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during Drosophila eye patterning. PLoS One 12(11): e0187571. PubMed ID: 29117266
Ubiquitination is a crucial post-translational modification that can target proteins for degradation. The E3 ubiquitin ligases are responsible for recognizing substrate proteins for ubiquitination, hence providing specificity to the process of protein degradation. This study describes a genetic modifier screen that identified E3 ligases that modified the rough-eye phenotype generated by expression of cindr RNAi transgenes during Drosophila eye development. In total, 36 E3 ligases, as well as 4 Cullins, were identified that modified the mild cindrRNA mis-patterning phenotype. This indicates possible roles for these E3s/Cullins in processes that require Cindr function, including cytoskeletal regulation, cell adhesion, cell signaling and cell survival. Three E3 ligases identified in this screen had previously been linked to regulating JNK signaling.
Forest, E., Logeay, R., Geminard, C., Kantar, D., Frayssinoux, F., Heron-Milhavet, L. and Djiane, A. (2018). The apical scaffold big Bang binds to spectrins and regulates the growth of Drosophila melanogaster wing discs. J Cell Biol. PubMed ID: 29326287
During development, cell numbers are tightly regulated, ensuring that tissues and organs reach their correct size and shape. Recent evidence has highlighted the intricate connections between the cytoskeleton and the regulation of the key growth control Hippo pathway. Looking for apical scaffolds regulating tissue growth, this study describes that Drosophila melanogaster big bang (Bbg), a poorly characterized multi-PDZ scaffold, controls epithelial tissue growth without affecting epithelial polarity and architecture. bbg-mutant tissues are smaller, with fewer cells that are less apically constricted than normal. Bbg binds to and colocalizes tightly with the beta-heavy-Spectrin/Kst subunit at the apical cortex and promotes Yki activity, F-actin enrichment, and the phosphorylation of the myosin II regulatory light chain Spaghetti squash. A model is proposed in which the spectrin cytoskeleton recruits Bbg to the cortex, where Bbg promotes actomyosin contractility to regulate epithelial tissue growth.

Monday, March 12th

Mondal, T., Bag, I., Sncvl, P., Garikapati, K. R., Bhadra, U. and Pal Bhadra, M. (2018). Two way controls of apoptotic regulators consign DmArgonaute-1 a better clasp on it. PLoS One 13(1): e0190548. PubMed ID: 29385168
The roles of rgonaute family role in mitotic cell cycle progression and apoptotic cell elimination are poorly understood. Ago-1 plays a role in cell cycle control related to G2/M cyclin in Drosophila. This study extends understanding the role of Ago-1 in regulating apoptosis during Drosophila development. JNK (c-Jun N-terminal kinase) pathway plays a crucial role in regulating apoptosis. This study has overexpressed Ago-1 in Drosophila eye and brain by employing UAS (upstream activation sequence)-GAL4 system under the expression of eye and brain specific driver. Overexpression of Ago-1 resulted in reduced number of ommatidia in the eye and produced smaller size brain in adult and larval Drosophila. A drastic reversal of the phenotype towards normal was observed upon introduction of a single copy of the dominant negative mutation of basket (bsk, Drosophila homolog of JNK) indicating an active and physical involvement of the bsk with Ago-1 in inducing developmental apoptotic process. Further study showed that Ago-1 stimulates phosphorylation of JNK through transforming growth factor-beta activated kinase 1- hemipterous (Tak1-hep) axis of JNK pathway. JNK phosphorylation results in upregulation of pro-apoptotic genes head involution defective (hid), grim and reaper (rpr) and induces activation of Drosophila caspases (cysteinyl aspartate; DRONC (Death regulator Nedd2-like caspase), ICE (alternatively Drice, Death related ICE-like caspase) and DCP1 (Death caspase-1) by inhibiting apoptotic inhibitor protein DIAP1 (Death-associated inhibitor of apoptosis 1). Further, Ago-1 also inhibits miR-14 expression to trigger apoptosis. These findings propose that Ago-1 acts as a key regulator in controlling cell death, tumor regression and stress response in metazoan providing a constructive bridge between RNAi machinery and cell death.
Lee, G., Kim, J., Kim, Y., Yoo, S. and Park, J. H. (2018). Identifying and monitoring neurons that undergo metamorphosis-regulated cell death (metamorphoptosis) by a neuron-specific caspase sensor (Casor) in Drosophila melanogaster. Apoptosis 23(1): 41-53. PubMed ID: 29224041
Activation of caspases is an essential step toward initiating apoptotic cell death. During metamorphosis of Drosophila melanogaster, many larval neurons are programmed for elimination to establish an adult central nervous system (CNS) as well as peripheral nervous system (PNS). However, their neuronal functions have remained mostly unknown due to the lack of proper tools to identify them. To obtain detailed information about the neurochemical phenotypes of the doomed larval neurons and their timing of death, a new GFP-based caspase sensor (Casor) was generated that is designed to change its subcellular position from the cell membrane to the nucleus following proteolytic cleavage by active caspases. Ectopic expression of Casor in vCrz and bursicon, two different peptidergic neuronal groups that had been well-characterized for their metamorphic programmed cell death, showed clear nuclear translocation of Casor in a caspase-dependent manner before their death. Similar events in some cholinergic neurons from both CNS and PNS. Moreover, Casor also reported significant caspase activities in the ventral and dorsal common excitatory larval motoneurons shortly after puparium formation. These motoneurons were previously unknown for their apoptotic fate. Unlike the events seen in the neurons, expression of Casor in non-neuronal cell types, such as glial cells and S2 cells, resulted in the formation of cytoplasmic aggregates, preventing its use as a caspase sensor in these cell types. Nonetheless, these results support Casor as a valuable molecular tool not only for identifying novel groups of neurons that become caspase-active during metamorphosis but also for monitoring developmental timing and cytological changes within the dying neurons.
Khandelwal, R., Sipani, R., Govinda Rajan, S., Kumar, R. and Joshi, R. (2017). Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS. PLoS Genet 13(10): e1007043. PubMed ID: 29023471
Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. Grh, AbdA, and Exd were found to function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. Exd function is independent of its known partner homothorax in this apoptosis. Some findings were extended to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. A mechanism is proposed where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.
Sarkar, S., Khatun, S., Dutta, M. and Roy, S. (2017). Trans-generational transmission of altered phenotype resulting from flubendiamide-induced changes in apoptosis in larval imaginal discs of Drosophila melanogaster. Environ Toxicol Pharmacol 56: 350-360. PubMed ID: 29121551
The eye and wing morphology of Drosophila maintain unique, stable pattern of genesis from eye and wing imaginal discs. Increased apoptosis in discs was found to be associated with flubendiamide (fluoride containing insecticide) exposure in Drosophila larvae. The chemical fed larvae on attaining adulthood revealed alterations in morphology and symmetry of their compound eyes and wings through scanning electron microscopy. Nearly 40% and 30% of flies (P generation) demonstrated alterations in eyes and wings respectively. Transmission electron microscopic study also established variation in the rhabdomere and pigment cell orientation as well as in the shape of the ommatidium. Subsequent SEM study with F1 and F2 generation flies also revealed structural variation in eye and wing. Decrease in percentage of altered eye and wing phenotype was noted in subsequent generations. Thus, flubendiamide was found to increase apoptosis in larvae and thereby cause morphological alteration in the adult Drosophila. This study further demonstrated trans-generational transmission of altered phenotype in three subsequent generations of Drosophila.
Sinenko, S. A. (2017). Proapoptotic function of deubiquitinase DUSP31 in Drosophila. Oncotarget 8(41): 70452-70462. PubMed ID: 29050293
Drosophila have been used to identify new components in apoptosis regulation. The Drosophila protein Dark forms an octameric apoptosome complex that induces the initiator caspase Dronc to trigger the caspase cell death pathway and, therefore, plays an important role in controlling apoptosis. Caspases and Dark are constantly expressed in cells, but their activity is blocked by DIAP1 E3 ligase-mediated ubiquitination and subsequent inactivation or proteasomal degradation. One of the regulatory mechanisms that stabilize proapoptotic factors is the removal of ubiquitin chains by deubiquitinases. A modified genetic screen for deubiquitinases (dsRNA lines) was performed to identify those involved in stabilizing proapoptotic components. Loss-of-function alleles of deubiquitinase DUSP31 were identified as suppressors of the Dronc overexpression phenotype. DUSP31 deficiency also suppresses apoptosis induced by the RHG protein, Grim. Genetic analysis revealed for the first time that DUSP31 deficiency sufficiently suppresses the Dark phenotype, indicating its involvement in the control of Dark/Dronc apoptosome function in invertebrate apoptosis.
Wang, Z., Xia, X., Yang, X., Zhang, X., Liu, Y., Wu, D., Fang, Y., Liu, Y., Xu, J., Qiu, Y. and Zhou, X. (2017). A picorna-like virus suppresses the N-end rule pathway to inhibit apoptosis. Elife 6. PubMed ID: 29231806
The N-end rule pathway is an evolutionarily conserved proteolytic system that degrades proteins containing N-terminal degradation signals called N-degrons, and has emerged as a key regulator of various processes. Viruses manipulate diverse host pathways to facilitate viral replication and evade antiviral defenses. However, it remains unclear if viral infection has any impact on the N-end rule pathway. In this study, using a picorna-like virus as a model, it was found that viral infection promoted the accumulation of caspase-cleaved Drosophila inhibitor of apoptosis 1 (DIAP1) by inducing the degradation of N-terminal amidohydrolase 1 (NTAN1), a key N-end rule component that identifies N-degron to initiate the process. The virus-induced NTAN1 degradation is independent of polyubiquitylation but dependent on proteasome. Furthermore, the virus-induced N-end rule pathway suppression inhibits apoptosis and benefits viral replication. Thus, these findings demonstrate that a virus can suppress the N-end rule pathway, and uncover a new mechanism for virus to evade apoptosis.

Friday, February 9th

Calpena, E., Lopez Del Amo, V., Chakraborty, M., Llamusi, B., Artero, R., Espinos, C. and Galindo, M. I. (2018). The Drosophila junctophilin gene is functionally equivalent to its four mammalian counterparts and is a modifier of a Huntingtin poly-Q expansion and the Notch pathway. Dis Model Mech 11(1). PubMed ID: 29208631
Members of the Junctophilin (JPH) protein family have emerged as key actors in all excitable cells, with crucial implications for human pathophysiology. In mammals, this family consists of four members (JPH1-JPH4) that are differentially expressed throughout excitable cells. The analysis of knockout mice lacking JPH subtypes has demonstrated their essential contribution to physiological functions in skeletal and cardiac muscles and in neurons. Moreover, mutations in the human JPH2 gene are associated with hypertrophic and dilated cardiomyopathies; mutations in JPH3 are responsible for the neurodegenerative Huntington's disease-like-2 (HDL2), whereas JPH1 acts as a genetic modifier in Charcot-Marie-Tooth 2K peripheral neuropathy. Drosophila melanogaster has a single junctophilin (jp) gene, as is the case in all invertebrates, which might retain equivalent functions of the four homologous JPH genes present in mammalian genomes. Therefore, owing to the lack of putatively redundant genes, a jp Drosophila model could provide an excellent platform to model the Junctophilin-related diseases, to discover the ancestral functions of the JPH proteins and to reveal new pathways. By up- and downregulation of Jp in a tissue-specific manner in Drosophila, this study shows that altering its levels of expression produces a phenotypic spectrum characterized by muscular deficits, dilated cardiomyopathy and neuronal alterations. Importantly, this study has demonstrated that Jp modifies the neuronal degeneration in a Drosophila model of Huntington's disease, and it has allowed uncovery of an unsuspected functional relationship with the Notch pathway. Therefore, this Drosophila model has revealed new aspects of Junctophilin function that can be relevant for the disease mechanisms of their human counterparts.
Rimkus, S. A. and Wassarman, D. A. (2018). A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant. PLoS One 13(1): e0190821. PubMed ID: 29338042
Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, was performed for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. Further studies of Ronnel were carried out because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.
Romey-Glusing, R., Li, Y., Hoffmann, J., von Frieling, J., Knop, M., Pfefferkorn, R., Bruchhaus, I., Fink, C. and Roeder, T. (2017). Nutritional regimens with periodically recurring phases of dietary restriction extend lifespan in Drosophila. Faseb J. PubMed ID: 29196499
Nutritional interventions such as caloric and dietary restriction increase lifespan in various animal models. To identify alternative and less demanding nutritional interventions that extend lifespan, fruit flies (Drosophila melanogaster) were subjected to weekly nutritional regimens that involved alternating a conventional diet with dietary restriction. Short periods of dietary restriction (up to 2 d) followed by longer periods of a conventional diet yielded minimal increases in lifespan. Three or more days of contiguous dietary restriction (DR) was necessary to yield a lifespan extension similar to that observed with persistent DR. Female flies were more responsive to these interventions than males. Physiologic changes known to be associated with prolonged DR, such as reduced metabolic rates, showed the same time course as lifespan extension. Moreover, concurrent transcriptional changes indicative of reduced insulin signaling were identified with DR. These physiologic and transcriptional changes were sustained, as they were detectable several days after switching to conventional diets. Taken together, diets with longer periods of DR extended lifespan concurrently with physiologic and transcriptional changes that may underlie this increase in lifespan.
Hussain, A., Pooryasin, A., Zhang, M., Loschek, L. F., La Fortezza, M., Friedrich, A. B., Blais, C. M., Ucpunar, H. K., Yepez, V. A., Lehmann, M., Gompel, N., Gagneur, J., Sigrist, S. J. and Grunwald Kadow, I. C. (2018). Inhibition of oxidative stress in cholinergic projection neurons fully rescues aging-associated olfactory circuit degeneration in Drosophila. Elife 7. PubMed ID: 29345616
Loss of the sense of smell is among the first signs of natural aging and neurodegenerative diseases such as Alzheimer's and Parkinson's. Cellular and molecular mechanisms promoting this smell loss are not understood. This study shows that Drosophila melanogaster also loses olfaction before vision with age. Within the olfactory circuit, cholinergic projection neurons show a reduced odor response accompanied by a defect in axonal integrity and reduction in synaptic marker proteins. Using behavioral functional screening, this study pinpoints that expression of the mitochondrial reactive oxygen scavenger SOD2 in cholinergic projection neurons is necessary and sufficient to prevent smell degeneration in aging flies. Together, these data suggest that oxidative stress induced axonal degeneration in a single class of neurons drives the functional decline of an entire neural network and the behavior it controls. Given the important role of the cholinergic system in neurodegeneration, the fly olfactory system could be a useful model for the identification of drug targets.
Hutson, R. L., Thompson, R. L., Bantel, A. P. and Tessier, C. R. (2018). Acamprosate rescues neuronal defects in the Drosophila model of Fragile X Syndrome. Life Sci. PubMed ID: 29317220
Several off-label studies have shown that acamprosate, a modulator of AMPA and GABAA receptors, can provide some clinical benefits in youth with Fragile X Syndrome (FXS), an autism spectrum disorder caused by loss of function of the highly conserved FMR1 gene. This study investigated the ability of acamprosate to rescue cellular, molecular and behavioral defects in the Drosophila model of FXS. A high (100mμM) and low (10mμM) dose of acamprosate was fed to Drosophila FXS (dfmr1 null) or genetic control (w1118) larvae and then analyzed in multiple paradigms. A larval crawling assay was used to monitor aberrant FXS behavior, overgrowth of the neuromuscular junction (NMJ) was quantified to assess neuronal development, and quantitative RT-PCR was used to evaluate expression of deregulated Calbindin 53E cbp53E mRNA. Acamprosate treatment partially or completely rescued all of the FXS phenotypes analyzed, according to dose. High doses rescued cellular overgrowth and dysregulated cbp53E mRNA expression, but aberrant crawling behavior was not affected. Low doses of acamprosate, however, did not affect synapse number at the NMJ, but could rescue NMJ overgrowth, locomotor defects, and cbp53E mRNA expression. This dual nature of acamprosate suggests multiple molecular mechanisms may be involved in acamprosate function depending on the dosage used. Acamprosate may be a useful therapy for FXS and potentially other autism spectrum disorders. However, understanding the molecular mechanisms involved with different doses of this drug will likely be necessary to obtain optimal results.
Sakakibara, Y., Sekiya, M., Fujisaki, N., Quan, X. and Iijima, K. M. (2018). Knockdown of wfs1, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in Drosophila. PLoS Genet 14(1): e1007196. PubMed ID: 29357349
Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic beta-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. This study investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases.
Lee, B. C., Lee, H. M., Kim, S., Avanesov, A. S., Lee, A., Chun, B. H., Vorbruggen, G. and Gladyshev, V. N. (2018). Expression of the methionine sulfoxide reductase lost during evolution extends Drosophila lifespan in a methionine-dependent manner. Sci Rep 8(1): 1010. PubMed ID: 29343716
Accumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met-R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. This study examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, this study exposes the potential of a combination of genetic and nutritional strategies in lifespan control.
Ordonez, D. G., Lee, M. K. and Feany, M. B. (2018). alpha-synuclein induces mitochondrial dysfunction through spectrin and the actin cytoskeleton. Neuron 97(1): 108-124.e106. PubMed ID: 29249285
Genetics and neuropathology strongly link alpha-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related alpha-synucleinopathies. This study describes a new Drosophila model of alpha-synucleinopathy based on widespread expression of wild-type human alpha-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant alpha-synuclein aggregation. Results of forward genetic screening and genetic analysis were used in this new model to demonstrate that alpha-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse alpha-synucleinopathy model and in postmortem brain tissue from patients with alpha-synucleinopathy. Importantly, evidence is provided that the interaction of alpha-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for alpha-synuclein induced neurodegeneration.
Jantrapirom, S., Lo Piccolo, L., Yoshida, H. and Yamaguchi, M. (2018) . A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities. Exp Cell Res 362(2): 461-471. PubMed ID: 29247619
Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. This study performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. The depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. The results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.
Steinkellner, T., Zell, V., Farino, Z. J., Sonders, M. S., Villeneuve, M., Freyberg, R. J., Przedborski, S., Lu, W., Freyberg, Z. and Hnasko, T. S. (2018). Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons. J Clin Invest 128(2): 774-788. PubMed ID: 29337309
Parkinson's disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. This study addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.

Thursday, March 8th

Farley, J. E., Burdett, T. C., Barria, R., Neukomm, L. J., Kenna, K. P., Landers, J. E. and Freeman, M. R. (2018). Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration. Proc Natl Acad Sci U S A. PubMed ID: 29295933
Genetic studies of Wallerian degeneration have led to the identification of signaling molecules (e.g., dSarm/Sarm1, Axundead, and Highwire) that function locally in axons to drive degeneration. This study identified a role for the Drosophila C2H2 zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in Drosophila glutamatergic sensory neurons results in either complete preservation of severed axons, or an axon death phenotype where axons fragment into large, continuous segments, rather than completely disintegrate. Peb is expressed in developing and mature sensory neurons, suggesting it is required to establish or maintain their competence to undergo axon death. peb mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with dsarm mutants, linking peb/RREB1 to the axon death signaling cascade. Surprisingly, Peb is only able to fully block axon death signaling in glutamatergic, but not cholinergic sensory neurons, arguing for genetic diversity in axon death signaling programs in different neuronal subtypes. These findings identify a transcription factor that regulates axon death signaling, and peb mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling.
Diesner, M., Predel, R. and Neupert, S. (2018). Neuropeptide mapping of Dimmed cells of adult Drosophila brain. J Am Soc Mass Spectrom. PubMed ID: 29372551
Neuropeptides are structurally highly diverse messenger molecules that act as regulators of many physiological processes such as development, metabolism, reproduction or behavior in general. Differentiation of neuropeptidergic cells often corresponds with the presence of the transcription factor Dimmed. In the central nervous system of the fruit fly Drosophila melanogaster, Dimmed commonly occurs in neuroendocrine neurons that release peptides as neurohormones but also in interneurons with complex branching patterns. Fly strains with green fluorescence protein (GFP)-expressing dimmed cells make it possible to systematically analyze the processed neuropeptides in these cells. This study mapped individual GFP-expressing neurons of adult D. melanogaster from the dimmed (c929)>GFP line. Using single cell mass spectrometry, 10 types of dimmed neurons from the brain/gnathal ganglion were examined. These cells included neuroendocrine cells with projection into the retrocerebral complex but also a number of large interneurons. Resulting mass spectra not only provided comprehensive data regarding mature products from 13 neuropeptide precursors but also evidence for the cellular co-localization of neuropeptides from different neuropeptide genes. The results can be implemented in a neuroanatomical map of the D. melanogaster brain.
Clemens, J., Ozeri-Engelhard, N. and Murthy, M. (2018). Fast intensity adaptation enhances the encoding of sound in Drosophila. Nat Commun 9(1): 134. PubMed ID: 29317624
To faithfully encode complex stimuli, sensory neurons should correct, via adaptation, for stimulus properties that corrupt pattern recognition. This study investigated sound intensity adaptation in the Drosophila auditory system, which is largely devoted to processing courtship song. Mechanosensory neurons (JONs) in the antenna are sensitive not only to sound-induced antennal vibrations, but also to wind or gravity, which affect the antenna's mean position. Song pattern recognition, therefore, requires adaptation to antennal position (stimulus mean) in addition to sound intensity (stimulus variance). Fast variance adaptation was discovered in Drosophila JONs that corrects for background noise over the behaviorally relevant intensity range. This study determined where mean and variance adaptation arises and how they interact. A computational model explains these results using a sequence of subtractive and divisive adaptation modules, interleaved by rectification. These results lay the foundation for identifying the molecular and biophysical implementation of adaptation to the statistics of natural sensory stimuli.
Donlea, J. M., Pimentel, D., Talbot, C. B., Kempf, A., Omoto, J. J., Hartenstein, V. and Miesenbock, G. (2018). Recurrent circuitry for balancing sleep need and sleep. Neuron 97(2): 378-389.e374. PubMed ID: 29307711
Sleep-promoting neurons in the dorsal fan-shaped body (dFB) of Drosophila are integral to sleep homeostasis, but how these cells impose sleep on the organism is unknown. This study reports that dFB neurons communicate via inhibitory transmitters, including allatostatin-A (AstA), with interneurons connecting the superior arch with the ellipsoid body of the central complex. These "helicon cells" express the galanin receptor homolog AstA-R1, respond to visual input, gate locomotion, and are inhibited by AstA, suggesting that dFB neurons promote rest by suppressing visually guided movement. Sleep changes caused by enhanced or diminished allatostatinergic transmission from dFB neurons and by inhibition or optogenetic stimulation of helicon cells support this notion. Helicon cells provide excitation to R2 neurons of the ellipsoid body, whose activity-dependent plasticity signals rising sleep pressure to the dFB. By virtue of this autoregulatory loop, dFB-mediated inhibition interrupts processes that incur a sleep debt, allowing restorative sleep to rebalance the books.
Gruntman, E., Romani, S. and Reiser, M. B. (2018). Simple integration of fast excitation and offset, delayed inhibition computes directional selectivity in Drosophila. Nat Neurosci. PubMed ID: 29311742
A neuron that extracts directionally selective motion information from upstream signals lacking this selectivity must compare visual responses from spatially offset inputs. Distinguishing among prevailing algorithmic models for this computation requires measuring fast neuronal activity and inhibition. In the Drosophila melanogaster visual system, a fourth-order neuron-T4-is the first cell type in the ON pathway to exhibit directionally selective signals. This study used in vivo whole-cell recordings of T4 to show that directional selectivity originates from simple integration of spatially offset fast excitatory and slow inhibitory inputs, resulting in a suppression of responses to the nonpreferred motion direction. A passive, conductance-based model of a T4 cell was constructed that accurately predicts the neuron's response to moving stimuli. These results connect the known circuit anatomy of the motion pathway to the algorithmic mechanism by which the direction of motion is computed.
Strother, J. A., Wu, S. T., Rogers, E. M., Eliason, J. L. M., Wong, A. M., Nern, A. and Reiser, M. B. (2018). Behavioral state modulates the ON visual motion pathway of Drosophila. Proc Natl Acad Sci U S A 115(1): E102-e111. PubMed ID: 29255026
The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. This study shows that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and this study shows that central octopaminergic neurons provide input to Mi4 and increase its excitability. It was further shown that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.

Wednesday, March 7th

Harbison, S. T., Serrano Negron, Y. L., Hansen, N. F. and Lobell, A. S. (2017). Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep. PLoS Genet 13(12): e1007098. PubMed ID: 29240764
Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. This study applied artificial selection for high and low night sleep duration to an outbred population of Drosophila melanogaster for 13 generations. Night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Whole genome sequence data revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and three of these genes and a larger genomic region were confirmed with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways-EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.
Kim, Y. K., Saver, M., Simon, J., Kent, C. F., Shao, L., Eddison, M., Agrawal, P., Texada, M., Truman, J. W. and Heberlein, U. (2018). Repetitive aggressive encounters generate a long-lasting internal state in Drosophila melanogaster males. Proc Natl Acad Sci U S A 115(5): 1099-1104. PubMed ID: 29339481
Multiple studies have investigated the mechanisms of aggressive behavior in Drosophila; however, little is known about the effects of chronic fighting experience. This study investigated if repeated fighting encounters would induce an internal state that could affect the expression of subsequent behavior. Wild-type males were trained to become winners or losers by repeatedly pairing them with hypoaggressive or hyperaggressive opponents, respectively. As described previously, it was observed that chronic losers tend to lose subsequent fights, while chronic winners tend to win them. Olfactory conditioning experiments showed that winning is perceived as rewarding, while losing is perceived as aversive. Moreover, the effect of chronic fighting experience generalized to other behaviors, such as gap-crossing and courtship. It is proposed that in response to repeatedly winning or losing aggressive encounters, male flies form an internal state that displays persistence and generalization; fight outcomes can also have positive or negative valence. Furthermore, it was shown that the activities of the PPL1-gamma1pedc dopaminergic neuron and the MBON-gamma1pedc>α/β mushroom body output neuron are required for aversion to an olfactory cue associated with losing fights.
Yap, M. H. W., Grabowska, M. J., Rohrscheib, C., Jeans, R., Troup, M., Paulk, A. C., van Alphen, B., Shaw, P. J. and van Swinderen, B. (2017). Oscillatory brain activity in spontaneous and induced sleep stages in flies. Nat Commun 8(1): 1815. PubMed ID: 29180766
Sleep is a dynamic process comprising multiple stages, each associated with distinct electrophysiological properties and potentially serving different functions. While these phenomena are well described in vertebrates, it is unclear if invertebrates have distinct sleep stages. Local field potential (LFP) recordings were performed on flies spontaneously sleeping, and their brain activity was compared to flies induced to sleep using either genetic activation of sleep-promoting circuitry or the GABAA agonist Gaboxadol. A transitional sleep stage was found associated with a 7-10 Hz oscillation in the central brain during spontaneous sleep. Oscillatory activity is also evident when sleep-promoting neurons were acutely activated in the dorsal fan-shaped body (dFB) of Drosophila. In contrast, sleep following Gaboxadol exposure is characterized by low-amplitude LFPs, during which dFB-induced effects are suppressed. Sleep in flies thus appears to involve at least two distinct stages: increased oscillatory activity, particularly during sleep induction, followed by desynchronized or decreased brain activity.
Takagi, S., Cocanougher, B. T., Niki, S., Miyamoto, D., Kohsaka, H., Kazama, H., Fetter, R. D., Truman, J. W., Zlatic, M., Cardona, A. and Nose, A. (2017). Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila. Neuron 96(6): 1373-1387.e1376. PubMed ID: 29198754
Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. This study investigated how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. A class of segmentally repeated second-order somatosensory interneurons, that was named Wave, was identified whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, premotor circuits were identified including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.
Mishra, A., Salari, A., Berigan, B. R., Miguel, K. C., Amirshenava, M., Robinson, A., Zars, B. C., Lin, J. L., Milescu, L. S., Milescu, M. and Zars, T. (2018). The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool. Sci Rep 8(1): 901. PubMed ID: 29343813
A good candidate for developing new thermogenetic tools is the Drosophila gustatory receptor family, which has been implicated in high-temperature avoidance behavior. Five members of the alternatively spliced Gr28b gene cluster were examined for temperature-dependent properties via three approaches: biophysical characterization in Xenopus oocytes, functional calcium imaging in Drosophila motor neurons, and behavioral assays in adult Drosophila. Gr28bD expression in Xenopus oocytes produced a non-specific cationic current that is activated by elevated temperatures. This current is non-inactivating and non-voltage dependent. When expressed in Drosophila motor neurons, Gr28bD can be used to change the firing pattern of individual cells in a temperature-dependent fashion. Pan-neuronal or motor neuron expression of Gr28bD can be used to alter fruit fly behavior with elevated temperatures. Together, these results validate the potential of the Gr28bD gene as a founding member of a new class of thermogenetic tools.
Shalaby, N. A., Pinzon, J. H., Narayanan, A. S., Jin, E. J., Ritz, M. P., Dove, R. J., Wolfenberg, H., Rodan, A. R., Buszczak, M. and Rothenfluh, A. (2018). JmjC domain proteins modulate circadian behaviors and sleep in Drosophila. Sci Rep 8(1): 815. PubMed ID: 29339751
Jumonji (JmjC) domain proteins (see Jarid2) are known regulators of gene expression and chromatin organization by way of histone demethylation. Chromatin modification and remodeling provides a means to modulate the activity of large numbers of genes, but the importance of this class of predicted histone-modifying enzymes for different aspects of post-developmental processes remains poorly understood. This study tested the function of all 11 non-lethal members in the regulation of circadian rhythms and sleep. Loss of every Drosophila JmjC gene affects different aspects of circadian behavior and sleep in a specific manner. Together these findings suggest that the majority of JmjC proteins function as regulators of behavior, rather than controlling essential developmental programs.

Tuesday, March 6th

Anding, A. L., Wang, C., Chang, T. K., Sliter, D. A., Powers, C. M., Hofmann, K., Youle, R. J. and Baehrecke, E. H. (2018). Vps13D encodes a ubiquitin-binding protein that is required for the regulation of mitochondrial size and clearance. Curr Biol 28(2): 287-295.e286. PubMed ID: 29307555
The clearance of mitochondria by autophagy, mitophagy, is important for cell and organism health, and known to be regulated by ubiquitin. During Drosophila intestine development, cells undergo a dramatic reduction in cell size and clearance of mitochondria that depends on autophagy, the E1 ubiquitin-activating enzyme Uba1, and ubiquitin. This study screen a collection of putative ubiquitin-binding domain-encoding genes for cell size reduction and autophagy phenotypes. The endosomal sorting complex required for transport (ESCRT) components TSG101 and Vps36, as well as the novel gene Vps13D were identified. Vps13D is an essential gene that is necessary for autophagy, mitochondrial size, and mitochondrial clearance in Drosophila. Interestingly, a similar mitochondrial phenotype is observed in VPS13D mutant human cells. The ubiquitin-associated (UBA) domain of Vps13D binds K63 ubiquitin chains, and mutants lacking the UBA domain have defects in mitochondrial size and clearance and exhibit semi-lethality, highlighting the importance of Vps13D ubiquitin binding in both mitochondrial health and development. VPS13D mutant cells possess phosphorylated DRP1 and mitochondrial fission factor (MFF) as well as DRP1 association with mitochondria, suggesting that VPS13D functions downstream of these known regulators of mitochondrial fission. In addition, the large Vps13D mitochondrial and cell size phenotypes are suppressed by decreased mitochondrial fusion gene function. Thus, these results provide a previously unknown link between ubiquitin, mitochondrial size regulation, and autophagy.
Blaquiere, J. A., Wong, K. K. L., Kinsey, S. D., Wu, J. and Verheyen, E. M. (2018). Homeodomain-interacting protein kinase promotes tumorigenesis and metastatic cell behavior. Dis Model Mech 11(1). PubMed ID: 29208636
Aberrations in signaling pathways that regulate tissue growth often lead to tumorigenesis. Homeodomain-interacting protein kinase (Hipk) family members are reported to have distinct and contradictory effects on cell proliferation and tissue growth. From these studies, it is clear that much remains to be learned about the roles of Hipk family protein kinases in proliferation and cell behavior. Previous work has shown that Drosophila Hipk is a potent growth regulator, thus it is predicted that it could have a role in tumorigenesis. In a study of Hipk-induced phenotypes, the formation of tumor-like structures was observed in multiple cell types in larvae and adults. Furthermore, elevated Hipk in epithelial cells induces cell spreading, invasion and epithelial-to-mesenchymal transition (EMT) in the imaginal disc. Further evidence comes from cell culture studies, in which Drosophila Hipk was expressed in human breast cancer cells, and it was shown to enhance proliferation and migration. Past studies have shown that Hipk can promote the action of conserved pathways implicated in cancer and EMT, such as Wnt/Wingless, Hippo, Notch and JNK. This study shows that Hipk phenotypes are not likely to arise from activation of a single target, but rather through a cumulative effect on numerous target pathways. Most Drosophila tumor models involve mutations in multiple genes, such as the well-known Ras(V12) model, in which EMT and invasiveness occur after the additional loss of the tumor suppressor gene scribble. This study reveals that elevated levels of Hipk on their own can promote both hyperproliferation and invasive cell behavior, suggesting that Hipk family members could be potent oncogenes and drivers of EMT.
Bala Tannan, N., Collu, G., Humphries, A. C., Serysheva, E., Weber, U. and Mlodzik, M. (2018). AKAP200 promotes Notch stability by protecting it from Cbl/lysosome-mediated degradation in Drosophila melanogaster. PLoS Genet 14(1): e1007153. PubMed ID: 29309414
AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. This study demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, AKAP200 was found to interact positively with Notch in both the eye and the thorax. It was further shown that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, this study demonstrated that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, these data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.
Zheng, Y., Liu, B., Wang, L., Lei, H., Pulgar Prieto, K. D. and Pan, D. (2017). Homeostatic control of Hpo/MST kinase activity through autophosphorylation-dependent recruitment of the STRIPAK PP2A phosphatase complex. Cell Rep 21(12): 3612-3623. PubMed ID: 29262338
The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. This study identifies a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities.
Ziegler, A. B., Thiele, C., Tenedini, F., Richard, M., Leyendecker, P., Hoermann, A., Soba, P. and Tavosanis, G. (2017). Cell-autonomous control of neuronal dendrite expansion via the fatty acid synthesis regulator SREBP. Cell Rep 21(12): 3346-3353. PubMed ID: 29262315
During differentiation, neurons require a high lipid supply for membrane formation as they elaborate complex dendritic morphologies. While glia-derived lipids support neuronal growth during development, the importance of cell-autonomous lipid production for dendrite formation has been unclear. Using Drosophila larva dendritic arborization (da) neurons, this study shows that dendrite expansion relies on cell-autonomous fatty acid production. The nociceptive class four (CIV) da neurons form particularly large space-filling dendrites. Dendrite formation in these CIVda neurons additionally requires functional sterol regulatory element binding protein (SREBP), a crucial regulator of fatty acid production. The dendrite simplification in srebp mutant CIVda neurons is accompanied by hypersensitivity of srebp mutant larvae to noxious stimuli. Taken together, this work reveals that cell-autonomous fatty acid production is required for proper dendritic development and establishes the role of SREBP in complex neurons for dendrite elaboration and function.
Hannaford, M. R., Ramat, A., Loyer, N. and Januschke, J. (2018). aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts. Elife 7. PubMed ID: 29364113
Cell fate assignment in the nervous system of vertebrates and invertebrates often hinges on the unequal distribution of molecules during progenitor cell division. This study addresses asymmetric fate determinant localization in the developing Drosophila nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. A step-wise polarization of Miranda occurs in larval neuroblasts, and it was found that Miranda's dynamics and cortical association are differently regulated between interphase and mitosis. In interphase, Miranda binds to the plasma membrane. Then, before nuclear envelope breakdown, Miranda is phosphorylated by aPKC and displaced into the cytoplasm. This clearance is necessary for the subsequent establishment of asymmetric Miranda localization. After nuclear envelope breakdown, actomyosin activity is required to maintain Miranda asymmetry. Therefore, phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization in neuroblasts.

Monday, March 5th

Bronk, P., Kuklin, E. A., Gorur-Shandilya, S., Liu, C., Wiggin, T. D., Reed, M. L., Marder, E. and Griffith, L. C. (2018). Regulation of Eag by calcium/calmodulin controls presynaptic excitability in Drosophila. J Neurophysiol [Epub ahead of print]. PubMed ID: 29364071
Drosophila Ether-a-go-go (Eag) is the founding member of a large family of voltage-gated K(+) channels, the KCNH family, which includes Kv10, 11 and 12. Concurrent binding of calcium/calmodulin (Ca(2+)/CaM) to N- and C-terminal sites inhibits mammalian EAG1 channels at sub-micromolar Ca(2+) concentrations, likely by causing pore constriction. Although the Drosophila EAG channel was believed to be Ca(2+)-insensitive, both the N- and C-terminal sites are conserved. This study shows that Drosophila EAG is inhibited by high Ca(2+) concentrations that are only present at plasma membrane Ca(2+) channel microdomains. To test the role of this regulation in vivo, mutations were engineered that block CaM-binding to the major C-terminal site of the endogenous eag locus, disrupting Ca(2+)-dependent inhibition. eag CaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca(2+) influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K(+) conductance. These results are predicted by a conductance-based multi-compartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca(2+) channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults (Bronk, 2018).
Bulgari, D., Jha, A., Deitcher, D. L. and Levitan, E. S. (2018). Myopic (HD-PTP, PTPN23) selectively regulates synaptic neuropeptide release. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 29378961
Neurotransmission is mediated by synaptic exocytosis of neuropeptide-containing dense-core vesicles (DCVs) and small-molecule transmitter-containing small synaptic vesicles (SSVs). Exocytosis of both vesicle types depends on Ca(2+) and shared secretory proteins. This study shows that increasing or decreasing expression of Myopic (mop, HD-PTP, PTPN23), a Bro1 domain-containing pseudophosphatase implicated in neuronal development and neuropeptide gene expression, increases synaptic neuropeptide stores at the Drosophila neuromuscular junction (NMJ). This occurs without altering DCV content or transport, but synaptic DCV number and age are increased. The effect on synaptic neuropeptide stores is accounted for by inhibition of activity-induced Ca(2+)-dependent neuropeptide release. cAMP-evoked Ca(2+)-independent synaptic neuropeptide release also requires optimal Myopic expression, showing that Myopic affects the DCV secretory machinery shared by cAMP and Ca(2+) pathways. Presynaptic Myopic is abundant at early endosomes, but interaction with the endosomal sorting complex required for transport III (ESCRT III) protein (CHMP4/Shrub) that mediates Myopic's effect on neuron pruning is not required for control of neuropeptide release. Remarkably, in contrast to the effect on DCVs, Myopic does not affect release from SSVs. Therefore, Myopic selectively regulates synaptic DCV exocytosis that mediates peptidergic transmission at the NMJ.
Bonello, T. T., Perez-Vale, K. Z., Sumigray, K. D. and Peifer, M. (2018). Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment. Development 145(2). PubMed ID: 29361565
Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. Defining the mechanisms underlying polarity establishment remains a key issue. Drosophila embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction positioning. This study defines mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane localization, recruitment to nascent junctions and macromolecular assembly at tricellular junctions. The data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy (PDZ-GEF) is crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe mediate adherens junction and tricellular junction recruitment but are dispensable for membrane localization. These data also support a role for Canoe multimerization. These multifactorial inputs shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. This study integrates the existing data into a new polarity establishment model.
Chen, R. and Swale, D. R. (2018). Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects. Sci Rep 8(1): 1617. PubMed ID: 29371678
A complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. The recent development of potent and selective small-molecule modulators of insect inward rectifier potassium (Kir) channels, Kir1, Kir2, or Kir3 in Drosophila, has enabled the interrogation of the physiological role and toxicological potential of Kir channels within various insect tissue systems. Therefore, this study aimed to highlight the physiological and functional role of neural Kir channels the central nervous system, muscular system, and neuromuscular system through pharmacological and genetic manipulations. The data provide significant evidence that Drosophila neural systems rely on the inward conductance of K(+) ions for proper function, since pharmacological inhibition and genetic ablation of neural Kir channels yielded dramatic alterations of the CNS spike discharge frequency and broadening and reduced amplitude of the evoked EPSP at the neuromuscular junction. Based on these data, it is concluded that neural Kir channels in insects (1) are critical for proper function of the insect nervous system, (2) represents an unexplored physiological pathway that is likely to shape the understanding of neuronal signaling, maintenance of membrane potentials, and maintenance of the ionic balance of insects, and (3) are capable of inducing acute toxicity to insects through neurological poisoning.
Wentzel, C., Delvendahl, I., Sydlik, S., Georgiev, O. and Muller, M. (2018). Dysbindin links presynaptic proteasome function to homeostatic recruitment of low release probability vesicles. Nat Commun 9(1): 267. PubMed ID: 29348419
This study explores the relationship between presynaptic homeostatic plasticity and proteasome function at the Drosophila neuromuscular junction. First, it was demonstrated that the induction of homeostatic plasticity is blocked after presynaptic proteasome perturbation. Proteasome inhibition potentiates release under baseline conditions but not during homeostatic plasticity, suggesting that proteasomal degradation and homeostatic plasticity modulate a common pool of vesicles. The vesicles that are regulated by proteasome function and recruited during homeostatic plasticity are highly EGTA sensitive, implying looser Ca2+ influx-release coupling. Similar to homeostatic plasticity, proteasome perturbation enhances presynaptic Ca2+ influx, readily-releasable vesicle pool size, and does not potentiate release after loss of specific homeostatic plasticity genes, including the schizophrenia-susceptibility gene dysbindin. Finally, genetic evidence is provided that Dysbindin levels regulate the access to EGTA-sensitive vesicles. Together, these data suggest that presynaptic protein degradation opposes the release of low-release probability vesicles that are potentiated during homeostatic plasticity and whose access is controlled by dysbindin.
Hauswirth, A. G., Ford, K. J., Wang, T., Fetter, R. D., Tong, A. and Davis, G. W. (2018). A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity. Elife 7. PubMed ID: 29303480
Presynaptic homeostatic plasticity stabilizes information transfer at synaptic connections in organisms ranging from insect to human. By analogy with principles of engineering and control theory, the molecular implementation of PHP is thought to require postsynaptic signaling modules that encode homeostatic sensors, a set point, and a controller that regulates transsynaptic negative feedback. The molecular basis for these postsynaptic, homeostatic signaling elements remains unknown. In this study, an electrophysiology-based screen of the Drosophila kinome and phosphatome defines a postsynaptic signaling platform that includes a required function for PI3K-cII, PI3K-cIII and the small GTPase Rab11 during the rapid and sustained expression of PHP. Evidence is presented that PI3K-cII localizes to Golgi-derived, clathrin-positive vesicles and is necessary to generate an endosomal pool of PI(3)P that recruits Rab11 to recycling endosomal membranes. A morphologically distinct subdivision of this platform concentrates postsynaptically where it is proposed to functions as a homeostatic controller for retrograde, trans-synaptic signaling.

Friday, March 2nd

Grmai, L., Hudry, B., Miguel-Aliaga, I. and Bach, E. A. (2018). Chinmo prevents transformer alternative splicing to maintain male sex identity. PLoS Genet 14(2): e1007203. PubMed ID: 29389999
Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. This feminization occurs by transcriptional and post-transcriptional regulation of traF. chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, this study shows that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. This work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.
Vimal, D., Kumar, S., Pandey, A., Sharma, D., Saini, S., Gupta, S., Ravi Ram, K. and Chowdhuri, D. K. (2017). Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation. Eur J Cell Biol. PubMed ID: 29290392
Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. This study analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1e00130 hypomorphic allele, this study reports female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility.
Tasnim, S. and Kelleher, E. S. (2017). p53 is required for female germline stem cell maintenance in P-element hybrid dysgenesis. Dev Biol [Epub ahead of print]. PubMed ID: 29294306
Hybrid dysgenesis is a sterility syndrome resulting from the mobilization of certain transposable elements in the Drosophila germline. Particularly extreme is the hybrid dysgenesis syndrome caused by P-element DNA transposons, in which dysgenic female ovaries often contain few or no germline cells. Those offspring that are produced from dysgenic germlines exhibit high rates of de novo mutation and recombination, implicating transposition-associated DNA damage as the cause of germline loss. However, how this loss occurs, in terms of the particular cellular response that is triggered (cell cycle arrest, senescence, or cell death) remains poorly understood. This study demonstrated that two components of the DNA damage response, Checkpoint kinase 2 and its downstream target p53, determine the frequency of ovarian atrophy that is associated with P-element hybrid dysgenesis. It was further shown that p53 is strongly induced in the germline stem cells (GSCs) of dysgenic females, and is required for their maintenance. These observations support the critical role for p53 in conferring tolerance of transposable element activity in stem cells.
Andreatta, G., Kyriacou, C. P., Flatt, T. and Costa, R. (2018). Aminergic signaling controls ovarian dormancy in Drosophila. Sci Rep 8(1): 2030. PubMed ID: 29391447
In response to adverse environmental conditions many organisms from nematodes to mammals deploy a dormancy strategy, causing states of developmental or reproductive arrest that enhance somatic maintenance and survival ability at the expense of growth or reproduction. Dormancy regulation has been studied in C. elegans and in several insects, but how neurosensory mechanisms act to relay environmental cues to the endocrine system in order to induce dormancy remains unclear. This fundamental question was examined in Drosophila by genetically manipulating aminergic neurotransmitter signaling in Drosophila melanogaster. Both serotonin and dopamine were found enhance adult ovarian dormancy, while the downregulation of their respective signaling pathways in endocrine cells or tissues (insulin producing cells, fat body, corpus allatum) reduces dormancy. In contrast, octopamine signaling antagonizes dormancy. These findings enhance understanding of the ability of organisms to cope with unfavorable environments and illuminate some of the relevant signaling pathways.
Tan, S. W. S., Yip, G. W., Suda, T. and Baeg, G. H. (2017). Small Maf functions in the maintenance of germline stem cells in the Drosophila testis. Redox Biol 15: 125-134. PubMed ID: 29245136
Reactive oxygen species (ROS) are byproducts generated during normal cellular metabolism, and redox states have been shown to influence stem cell self-renewal and lineage commitment across phyla. However, the downstream effectors of ROS signaling that control stem cell behavior remain largely unexplored. This study used the Drosophila testis as an in vivo model to identify ROS-induced effectors that are involved in the differentiation process of germline stem cells (GSCs). In the Affymetrix microarray analysis, 152 genes were either upregulated or downregulated during GSC differentiation induced by elevated levels of ROS, and a follow-up validation of the gene expression by qRT-PCR showed a Spearman's rho of 0.9173 (P<0.0001). Notably, 47 (31%) of the identified genes had no predicted molecular function or recognizable protein domain. These suggest the robustness of this microarray analysis, which identified many uncharacterized genes, possibly with an essential role in ROS-induced GSC differentiation. maf-S was shown to be transcriptionally downregulated by oxidative stress, and maf-S knockdown promotes GSC differentiation, but Maf-S overexpression conversely results in an over-growth of GSC-like cells by promoting the mitotic activity of germ cell lineage. Together with the facts that Maf-S regulates ROS levels and genetically interacts with Keap1/Nrf2 in GSC maintenance, this study suggests that Maf-S plays an important role in the Drosophila testis GSC maintenance by participating in the regulation of redox homeostasis.
Chen, D., Zhou, L., Sun, F., Sun, M. and Tao, X. (2018). Cyclin B3 deficiency impairs germline stem cell maintenance and its overexpression delays cystoblast differentiation in Drosophila ovary. Int J Mol Sci 19(1). PubMed ID: 29351213
It is well known that cyclinB3 (cycB3) plays a key role in the control of cell cycle progression. However, whether cycB3 is involved in stem cell fate determination remains unknown. The Drosophila ovary provides an exclusive model for studying the intrinsic and extrinsic factors that modulate the fate of germline stem cells (GSCs). Here, using this model, Drosophila cycB3 was shown to plays a new role in controlling the fate of germline stem cells (GSC). Results from cycB3 genetic analyses demonstrate that cycB3 is intrinsically required for GSC maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that cycB3 is not involved in Dad-mediated regulation of Bmp signaling, or required for dpp-induced bam transcriptional silencing. Double mutants of bam and cycB3 phenocopied bam single mutants, suggesting that cycB3 functions in a bam-dependent manner in GSCs. Deficiency of cycB3 fails to cause apoptosis in GSCs or influence cystoblast (CB) differentiation into oocytes. Furthermore, overexpression of cycB3 dramatically increases the CB number in Drosophila ovaries, suggesting that an excess of cycB3 function delays CB differentiation. Given that the cycB3 gene is evolutionarily conserved, from insects to humans, cycB3 may also be involved in controlling the fate of GSCs in humans.
Feng, L., Shi, Z., Xie, J., Ma, B. and Chen, X. (2018). Enhancer of polycomb maintains germline activity and genome integrity in Drosophila testis. Cell Death Differ [Epub ahead of print]. PubMed ID: 29362481
proliferation and differentiation, as well as ensure DNA damage repair. This study used the Drosophila male germline stem cell system to study how a chromatin factor, enhancer of polycomb [E(Pc)], regulates the proliferation-to-differentiation (mitosis-to-meiosis) transition and DNA damage repair. Two critical targets of E(Pc) were identified. First, E(Pc) represses CycB transcription, likely through modulating H4 acetylation. Second, E(Pc) is required for accumulation of an important germline differentiation factor, Bag-of-marbles (Bam), through post-transcriptional regulation. When E(Pc) is downregulated, increased CycB and decreased Bam are both responsible for defective mitosis-to-meiosis transition in the germline. Moreover, DNA double-strand breaks (DSBs) accumulate upon germline inactivation of E(Pc) under both physiological condition and recovery from heat shock-induced endonuclease expression. Failure of robust DSB repair likely leads to germ cell loss. Finally, compromising the activity of Tip60, a histone acetyltransferase, leads to germline defects similar to E(Pc) loss-of-function, suggesting that E(Pc) acts cooperatively with Tip60. Together, these data demonstrate that E(Pc) has pleiotropic roles in maintaining male germline activity and genome integrity. These findings will help elucidate the in vivo molecular mechanisms of E(Pc).
Singh, A., Dutta, D., Paul, M. S., Verma, D., Mutsuddi, M. and Mukherjee, A. (2018). Pleiotropic functions of the chromodomain-containing protein Hat-trick during oogenesis in Drosophila melanogaster. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 29367451
Chromatin-remodeling proteins play a profound role in the transcriptional regulation of gene expression during development. This study shows that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome and that a mild expression is observed in follicle cells. Co-localization of Hat-trick with Heterochromatin Protein 1 and a Synaptonemal Complex component- C(3)G along with the diffused karyosome after hat-trick down-regulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. Germline mosaic analysis reveals that hat-trick is required for maintaining the dorso-ventral patterning of eggs by regulating the expression of Gurken. The increased incidence of Double strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility and consequently misexpression and altered localization of Gurken in hat-trick mutant egg chambers, clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, it is speculated that hat-trick plays a substantial role in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. These results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster.

Thursday, March 1st

Caria, S., Magtoto, C. M., Samiei, T., Portela, M., Lim, K. Y. B., How, J. Y., Stewart, B. Z., Humbert, P. O., Richardson, H. E. and Kvansakul, M. (2018). Drosophila melanogaster Guk-holder interacts with the Scribbled PDZ1 domain and regulates epithelial development with Scribbled and Discs Large. J Biol Chem [Epub ahead of print]. PubMed ID: 29378849
Epithelial cell polarity is controlled by components of the Scribble polarity module, and its regulation is critical for tissue architecture and cell proliferation and cell migration. In Drosophila melanogaster, the adaptor protein Guk-holder (Gukh) binds to the Scribbled (Scrib) and Discs Large (Dlg) components of the Scribble polarity module and plays an important role in the formation of neuromuscular junctions. However, Gukhs role in epithelial tissue formation and the molecular basis for the Scrib-Gukh interaction remain to be defined. This study shows using isothermal titration calorimetry that the Scrib PDZ1 domain is the major site for an interaction with Gukh. Furthermore, the structural basis of this interaction is defined by determining the crystal structure of the Scrib PDZ1-Gukh complex. The C-terminal PDZ-binding motif of Gukh is located in the canonical ligand binding groove of Scrib PDZ1, and utilizes an unusually extensive network of hydrogen bonds and ionic interactions to enable binding to PDZ1 with high affinity. The roles of Gukh along with those of Scrib and Dlg were examined in Drosophila epithelial tissues, and found Gukh was found to be expressed in larval-wing and eye-epithelial tissues and co-localizes with Scrib and Dlg at the apical cell cortex. Importantly, it was shown that Gukh functions with Scrib and Dlg in the development of Drosophila epithelial tissues, with depletion of Gukh enhancing the eye- and wing-tissue defects caused by Scrib or Dlg depletion. Overall these findings reveal that Scribs PDZ1 domain functions in the interaction with Gukh and that the Scrib-Gukh interaction has a key role in epithelial tissue development in Drosophila.
Hamm, D. C., Larson, E. D., Nevil, M., Marshall, K. E., Bondra, E. R. and Harrison, M. M. (2017). A conserved maternal-specific repressive domain in Zelda revealed by Cas9-mediated mutagenesis in Drosophila melanogaster. PLoS Genet 13(12): e1007120. PubMed ID: 29261646
In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome becomes transcriptionally active hours after fertilization. Transcriptional activation during this maternal-to-zygotic transition (MZT) is tightly coordinated with the degradation of maternally provided mRNAs. In Drosophila melanogaster, the transcription factor Zelda plays an essential role in widespread activation of the zygotic genome. While Zelda expression is required both maternally and zygotically, the mechanisms by which it functions to remodel the embryonic genome and prepare the embryo for development remain unclear. Using Cas9-mediated genome editing to generate targeted mutations in the endogenous zelda locus, this study determined the functional relevance of protein domains conserved amongst Zelda orthologs. A highly conserved zinc-finger domain was identified that is essential for the maternal, but not zygotic functions of Zelda. Animals homozygous for mutations in this domain survived to adulthood, but embryos inheriting these loss-of-function alleles from their mothers died late in embryogenesis. These mutations did not interfere with the capacity of Zelda to activate transcription in cell culture. Unexpectedly, these mutations generated a hyperactive form of the protein and enhanced Zelda-dependent gene expression. These data have defined a protein domain critical for controlling Zelda activity during the MZT, but dispensable for its roles later in development, for the first time separating the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are required for establishing the developmental program during the MZT. It is proposed that tightly regulated gene expression is essential to navigate the MZT and that failure to precisely execute this developmental program leads to embryonic lethality.
Das, S. and Knust, E. (2018). A dual role of the extracellular domain of Drosophila Crumbs for morphogenesis of the embryonic neuroectoderm. Biol Open 7(1). PubMed ID: 29374056
Epithelia are highly polarised tissues and several highly conserved polarity protein complexes serve to establish and maintain polarity. The transmembrane protein Crumbs (Crb), the central component of the Crb protein complex, is required, among others, for the maintenance of polarity in most epithelia in the Drosophila embryo. However, different epithelia exhibit different phenotypic severity upon loss of crb. Using a transgenomic approach allowed the more accurate definition of the role of crb in different epithelia. In particular, evidence is provided that the loss of epithelial tissue integrity in the ventral epidermis of crb mutant embryos is due to impaired actomyosin activity and an excess number of neuroblasts. It was demonstrated that the intracellular domain of Crb could only partially rescue this phenotype, while it is able to completely restore tissue integrity in other epithelia. Based on these results, a dual role of the extracellular domain of Crb in the ventral neuroectoderm is suggested. First, it is required for apical enrichment of the Crb protein, which in turn regulates actomyosin activity and thereby ensures tissue integrity; and second, the extracellular domain of Crb stabilises the Notch receptor and thereby ensures proper Notch signalling and specification of the correct number of neuroblasts.
Xiang, J., Reding, K., Heffer, A. and Pick, L. (2017). Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle, Dermestes maculatus. Development 144(24):4625-4636. PubMed ID: 29084804
Evolutionary Homolog Study
A set of pair-rule segmentation genes (PRGs) promote the formation of alternate body segments in Drosophila melanogaster While Drosophila embryos are long-germ, with segments specified more-or-less simultaneously, most insects add segments sequentially as the germband elongates. The hide beetle, Dermestes maculatus, represents an intermediate between short- and long-germ development, ideal for comparative study of PRGs. This study shows that eight of nine Drosophila PRG-orthologs are expressed in stripes in Dermestes. Functional results parse these genes into three groups: Dmac-eve, -odd, and -run play roles in both germband elongation and PR-patterning. Dmac-slp and -prd function exclusively as complementary, classic PRGs, supporting functional decoupling of elongation and segment formation. Orthologs of ftz, ftz-f1, h, and opa show more variable function in Dermestes and other species. While extensive cell death generally prefigured Dermestes PRG RNAi cuticle defects, an organized region with high mitotic activity near the margin of the segment addition zone likely contributes to truncation of eve(RNAi) embryos. These results suggest general conservation of clock-like regulation of PR-stripe addition in sequentially-segmenting species while highlighting regulatory re-wiring involving a subset of PRG-orthologs.
Sanchez-Sanchez, B. J., Urbano, J. M., Comber, K., Dragu, A., Wood, W., Stramer, B. and Martin-Bermudo, M. D. (2017). Drosophila embryonic hemocytes produce laminins to strengthen migratory response. Cell Rep 21(6): 1461-1470. PubMed ID: 29117553
The most prominent developmental function attributed to the extracellular matrix (ECM) is cell migration. While cells in culture can produce ECM to migrate, the role of ECM in regulating developmental cell migration is classically viewed as an exogenous matrix presented to the moving cells. In contrast to this view, this study shows that Drosophila embryonic hemocytes deposit their own laminins in streak-like structures to migrate efficiently throughout the embryo. With the help of transplantation experiments, live microscopy, and image quantification, it was demonstrated that autocrine-produced laminin regulates hemocyte migration by controlling lamellipodia dynamics, stability, and persistence. Proper laminin deposition is regulated by the RabGTPase Rab8, which is highly expressed and required in hemocytes for lamellipodia dynamics and migration. These results thus support a model in which, during embryogenesis, the Rab8-regulated autocrine deposition of laminin reinforces directional and effective migration by stabilizing cellular protrusions and strengthening otherwise transient adhesion states.
Requena, D., Alvarez, J. A., Gabilondo, H., Loker, R., Mann, R. S. and Estella, C. (2017). Origins and Specification of the Drosophila Wing. Curr Biol 27(24): 3826-3836.e3825. PubMed ID: 29225023

Two main hypotheses have been proposed for the origin of the insect wing: the paranotal hypothesis, which suggests that wings evolved as an extension of the dorsal thorax, and the gill-exite hypothesis, which proposes that wings were derived from a modification of a pre-existing branch at the dorsal base (subcoxa) of the leg. This study addresses this question by studying how wing fates are initially specified during Drosophila embryogenesis, by characterizing a cis-regulatory module (CRM) from the snail (sna) gene, sna-DP (for dorsal primordia). sna-DP specifically marks the early primordia for both the wing and haltere, collectively referred to as the DP. The inputs that activate sna-DP are distinct from those that activate Distalless, a marker for leg fates. Further, in genetic backgrounds in which the leg primordia are absent, the DP are still partially specified. However, lineage-tracing experiments demonstrate that cells from the early leg primordia contribute to both ventral and dorsal appendage fates. Together, these results suggest that the wings of Drosophila have a dual developmental origin: two groups of cells, one ventral and one more dorsal, give rise to the mature wing. It is suggested that the dual developmental origins of the wing may be a molecular remnant of the evolutionary history of this appendage, in which cells of the subcoxa of the leg coalesced with dorsal outgrowths to evolve a dorsal appendage with motor control.

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