What's hot today
Thursday, July 31st, 2014
Butts, T., Hanzel, M. and Wingate, R. J. (2014). Transit amplification in the amniote cerebellum evolved via a heterochronic shift in NeuroD1 expression. Development 141: 2791-2795. PubMed ID: 25005474
The cerebellum has evolved elaborate foliation in the amniote lineage as a consequence of extensive Atoh1-mediated transit amplification in an external germinal layer (EGL) comprising granule cell precursors. To explore the evolutionary origin of this layer, the molecular geography of cerebellar development was examined throughout the life cycle of Xenopus laevis. At metamorphic stages Xenopus displays a superficial granule cell layer that is not proliferative and expresses both Atoh1 (see Drosophila Atonal) and NeuroD1 (see Drosophila Tap), a marker of postmitotic cerebellar granule cells. Premature misexpression of NeuroD1 in chick partially recapitulates the amphibian condition by suppressing transit amplification. However, unlike in the amphibian, granule cells fail to enter the EGL. Furthermore, misexpression of NeuroD1 once the EGL is established both triggers radial migration and downregulates Atoh1. These results show that the evolution of transit amplification in the EGL required adaptation of NeuroD1, both in the timing of its expression and in its regulatory function, with respect to Atoh1.
Cambier, L., Plate, M., Sucov, H. M. and Pashmforoush, M. (2014). Nkx2-5 regulates cardiac growth through modulation of Wnt signaling by R-spondin3. Development 141: 2959-2971. PubMed ID: 25053429
A complex regulatory network of morphogens and transcription factors is essential for normal cardiac development. Nkx2-5 (See Drosophila Tinman) is among the earliest known markers of cardiac mesoderm that is central to the regulatory pathways mediating second heart field (SHF) development. This study has examined the specific requirements for Nkx2-5 in the SHF progenitors. Nkx2-5 was found to potentiate Wnt signaling by regulating the expression of the R-spondin3 (Rspo3) gene during cardiogenesis. R-spondins are secreted factors and potent Wnt agonists that in part regulate stem cell proliferation. The data show that Rspo3 is markedly downregulated in Nkx2-5 mutants and that Rspo3 expression is regulated by Nkx2-5. Conditional inactivation of Rspo3 in the Isl1 lineage resulted in embryonic lethality secondary to impaired development of SHF. More importantly, it was found that Wnt signaling is significantly attenuated in Nkx2-5 mutants and that enhancing Wnt/beta-catenin signaling by pharmacological treatment or by transgenic expression of Rspo3 rescues the SHF defects in the conditional Nkx2-5(+/-) mutants. A previously unrecognized genetic link between Nkx2-5 and Wnt signaling was uncovered that supports continued cardiac growth and proliferation during development. Identification of Rspo3 in cardiac development provides a new paradigm in temporal regulation of Wnt signaling by cardiac-specific transcription factors.
Zalc, A., Hayashi, S., Aurade, F., Brohl, D., Chang, T., Mademtzoglou, D., Mourikis, P., Yao, Z., Cao, Y., Birchmeier, C. and Relaix, F. (2014). Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest. Development 141: 2780-2790. PubMed ID: 25005473
A central question in development is to define how the equilibrium between cell proliferation and differentiation is temporally and spatially regulated during tissue formation. This study addresses how interactions between cyclin-dependent kinase inhibitors essential for myogenic growth arrest (p21cip1 and p57kip2; see Drosophila Dacapo), the Notch pathway and myogenic regulatory factors (MRFs; see for example Drosophila Nautilus) orchestrate the proliferation, specification and differentiation of muscle progenitor cells. It was first shown that cell cycle exit and myogenic differentiation can be uncoupled. In addition, it was establish that skeletal muscle progenitor cells require Notch signaling to maintain their cycling status. Using several mouse models combined with ex vivo studies, it was demonstrated that Notch signaling is required to repress p21cip1 and p57kip2 expression in muscle progenitor cells. Finally, a muscle-specific regulatory element of p57kip2 directly activated by MRFs was identified in myoblasts but was found to be repressed by the Notch targets Hes1/Hey1 (see Drosophila Hairy) in progenitor cells. A molecular mechanism is proposed whereby information provided by Hes/Hey downstream of Notch as well as MRF activities are integrated at the level of the p57kip2 enhancer to regulate the decision between progenitor cell maintenance and muscle differentiation.
Suda, N., Itoh, T., Nakato, R., Shirakawa, D., Bando, M., Katou, Y., Kataoka, K., Shirahige, K., Tickle, C. and Tanaka, M. (2014). Dimeric combinations of MafB, cFos and cJun control the apoptosis-survival balance in limb morphogenesis. Development 141: 2885-2894. PubMed ID: 25005477
Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. This study showed that MafB (see Drosophila Traffic jam) was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos (see Drosophila Kayak) heterodimers repressed apoptosis, whereas MafB/cJun (see Drosophila Jun) heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. The data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73.
Wednesday, July 30th
Anani, S., Bhat, S., Honma-Yamanaka, N., Krawchuk, D. and Yamanaka, Y. (2014). Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre-implantation mouse embryo. Development 141: 2813-2824. PubMed ID: 24948601
In the mouse embryo, asymmetric divisions during the 8-16 cell division generate two cell types, polar and apolar cells, that are allocated to outer and inner positions, respectively. This outer/inner configuration is the first sign of the formation of the first two cell lineages: trophectoderm (TE) and inner cell mass (ICM). Outer polar cells become TE and give rise to the placenta, whereas inner apolar cells become ICM and give rise to the embryo proper and yolk sac. This study analyzed the frequency of asymmetric divisions during the 8-16 cell division and assessed the relationships between cell polarity, cell and nuclear position, and Hippo signaling activation, the pathway that initiates lineage-specific gene expression in 16-cell embryos. Although the frequency of asymmetric divisions varied in each embryo, it was found that more than six blastomeres divided asymmetrically in most embryos. Interestingly, many apolar cells in 16-cell embryos were located at outer positions, whereas only one or two apolar cells were located at inner positions. Live imaging analysis showed that outer apolar cells were eventually internalized by surrounding polar cells. Using isolated 8-cell blastomeres, the internalization process of apolar cells was carefully analyzed, and indications were found of higher cortical tension in apolar cells than in polar cells. Last, apolar cells were found to activate Hippo signaling prior to taking inner positions. These results suggest that polar and apolar cells have intrinsic differences that establish outer/inner configuration and differentially regulate Hippo signaling to activate lineage-specific gene expression programs.
Kumar, S. and Duester, G. (2014). Retinoic acid controls body axis extension by directly repressing Fgf8 transcription. Development 141: 2972-2977. PubMed ID: 25053430
Retinoic acid (RA) generated in the mesoderm of vertebrate embryos controls body axis extension by downregulating Fgf8 (see Drosophila Pyramus and Thisbe) expression in cells exiting the caudal progenitor zone. RA activates transcription by binding to nuclear RA receptors (RARs) at RA response elements (RAREs), but it is unknown whether RA can directly repress transcription. This study analyzed a conserved RARE upstream of Fgf8 that binds RAR isoforms in mouse embryos. Transgenic embryos carrying Fgf8 fused to lacZ exhibited expression similar to caudal Fgf8, but deletion of the RARE resulted in ectopic trunk expression extending into somites and neuroectoderm. Epigenetic analysis using chromatin immunoprecipitation of trunk tissues from E8.25 wild-type and Raldh2(-/-) embryos lacking RA synthesis revealed RA-dependent recruitment of the repressive histone marker H3K27me3 (see Drosophila H3) and polycomb repressive complex 2 (PRC2) near the Fgf8 RARE. The co-regulator RERE, the loss of which results in ectopic Fgf8 expression and somite defects, was recruited near the RARb RARE by RA, but was released from the Fgf8 RARE by RA. These findings demonstrate that RA directly represses Fgf8 through a RARE-mediated mechanism that promotes repressive chromatin, thus providing valuable insight into the mechanism of RA-FGF antagonism during progenitor cell differentiation.
Michailovici, I., Harrington, H. A., Azogui, H. H., Yahalom-Ronen, Y., Plotnikov, A., Ching, S., Stumpf, M. P., Klein, O. D., Seger, R. and Tzahor, E. (2014). Nuclear to cytoplasmic shuttling of ERK promotes differentiation of muscle stem/progenitor cells. Development 141: 2611-2620. PubMed ID: 24924195
The transition between the proliferation and differentiation of progenitor cells is a key step in organogenesis, and alterations in this process can lead to developmental disorders. The extracellular signal-regulated kinase 1/2 (ERK) signaling pathway is one of the most intensively studied signaling mechanisms that regulates both proliferation and differentiation. How a single molecule (e.g. ERK; see Drosophila Rolled) can regulate two opposing cellular outcomes is still a mystery. Using both chick and mouse models, this study shed light on the mechanism responsible for the switch from proliferation to differentiation of head muscle progenitors and implicate ERK subcellular localization. Manipulation of the fibroblast growth factor (FGF)-ERK signaling pathway in chick embryos in vitro and in vivo demonstrated that blockage of this pathway accelerated myogenic differentiation, whereas its activation diminished it. Next, whether the spatial subcellular localization of ERK could act as a switch between proliferation (nuclear ERK) and differentiation (cytoplasmic ERK) of muscle progenitors was examined. A myristoylated peptide that blocks importin 7-mediated ERK nuclear translocation induced robust myogenic differentiation of muscle progenitor/stem cells in both head and trunk. In the mouse, analysis of Sprouty (see Drosophila Sprouty) mutant embryos revealed that increased ERK signaling suppressed both head and trunk myogenesis. These findings, corroborated by mathematical modeling, suggest that ERK shuttling between the nucleus and the cytoplasm provides a switch-like transition between proliferation and differentiation of muscle progenitors.
Schilling, S., Steiner, S., Zimmerli, D. and Basler, K. (2014). A regulatory receptor network directs the range and output of the Wingless signal. Development 141: 2483-2493. PubMed ID: 24917503
The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg - Arrow (Arr) and Frizzled 2 (Fz2) - are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. This study analyzed the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. These experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by an in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, a 200 bp regulatory element was identified in the arr locus that can account for the Arr gradient, and this was shown to be indirectly negatively controlled by Wg activity. Finally, the role of Frizzled 3 (Fz3) was examined in this system, and its expression, which is induced by Wg, was found to contribute to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, these results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system.
Tuesday, July 29th
Singh, P., Ramdas Nair, A. and Cabernard, C. (2014). The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts. Curr Biol [Epub ahead of print]. PubMed ID: 24954048
Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates. Drosophila neuroblasts, the neural precursors of the fly's central nervous system, contain molecularly and physically asymmetric centrosomes, established through differences in pericentriolar matrix (PCM) retention. For instance, the daughter centriole maintains PCM and thus microtubule-organizing center (MTOC) activity through Polo-mediated phosphorylation of Centrobin (Cnb). The mother centriole, however, quickly downregulates PCM and moves away from the apical cortex, randomly migrating through the cytoplasm until maturation sets in at prophase. How PCM downregulation is molecularly controlled is currently unknown, but it involves Pericentrin (PCNT)-like protein (PLP) to prevent premature Polo localization and thus MTOC activity. This study report that the centriolar protein Bld10, the fly ortholog of Cep135, is required to establish centrosome asymmetry in Drosophila neuroblasts through shedding of Polo from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM, generating two active, improperly positioned MTOCs. Failure to shed Polo and PCM causes spindle alignment and centrosome segregation defects, resulting in neuroblasts incorrectly retaining the older mother centrosome. Since Cep135 is implicated in primary microcephaly, it is speculated that perturbed centrosome asymmetry could contribute to this rare neurodevelopmental disease.
Galletta, B. J., Guillen, R. X., Fagerstrom, C. J., Brownlee, C. W., Lerit, D. A., Megraw, T. L., Rogers, G. C. and Rusan, N. M. (2014). Drosophila Pericentrin requires interaction with Calmodulin for its function at centrosomes and neuronal basal bodies, but not at sperm basal bodies. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25031429
Pericentrin is a critical centrosomal protein required for organizing pericentriolar material (PCM) in mitosis. Mutations in Pericentrin cause the human genetic disorder MOPD II, making a detailed understanding of its regulation extremely important. Germaine to Pericentrin's function in organizing PCM is its ability to localize to the centrosome through the conserved C-terminal PACT domain. This study used Drosophila Pericentrin-Like-Protein (PLP) to understand how the PACT domain is regulated. The interaction of PLP with Calmodulin (CaM) at two highly conserved CaM binding sites in the PACT domain was shown to control the proper targeting of PLP to the centrosome. Disrupting the PLP-CaM interaction with single point mutations renders PLP inefficient in localizing to centrioles in cultured S2 cells and Drosophila neuroblasts. Although levels of PCM are unaffected, it is highly disorganized. Basal body formation was also demonstrated in the male testes and the production of functional sperm does not rely on the PLP-CaM interaction, while production of functional mechanosensory neurons does.
Liu, Q. X., Wang, X. F., Ikeo, K., Hirose, S., Gehring, W. J. and Gojobori, T. (2014). Evolutionarily conserved transcription factor Apontic controls the G1/S progression by inducing cyclin E during eye development. Proc Natl Acad Sci U S A 111: 9497-9502. PubMed ID: 24979795
During Drosophila eye development, differentiation initiates in the posterior region of the eye disk and progresses anteriorly as a wave marked by the morphogenetic furrow (MF), which demarcates the boundary between anterior undifferentiated cells and posterior differentiated photoreceptors. However, the mechanism underlying the regulation of gene expression immediately before the onset of differentiation remains unclear. This study shows that Apontic (Apt), which is an evolutionarily conserved transcription factor, is expressed in the differentiating cells posterior to the MF. Moreover, it directly induces the expression of cyclin E and is also required for the G1-to-S phase transition, which is known to be essential for the initiation of cell differentiation at the MF. These observations identify a pathway crucial for eye development, governed by a mechanism in which Cyclin E promotes the G1-to-S phase transition when regulated by Apt.
Spiro, Z., Thyagarajan, K., De Simone, A., Trager, S., Afshar, K. and Gonczy, P. (2014). Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos. Development 141: 2712-2723. PubMed ID: 24961801
Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). This paper reports that the clathrin heavy chain CHC-1 (see Drosophila Clathrin) negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. A similar role was established for the cytokinesis/apoptosis/RNA-binding protein CAR-1, and it was uncovered that CAR-1 is needed to maintain proper levels of CHC-1. CHC-1 was shown to be necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, it was established that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1RNAi embryos. A 2D computational model was developed to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, these findings lead to a proposal that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension.
Monday, July 28th
Xie, G., Yu, Z., Jia, D., Jiao, R. and Deng, W. M. (2014). E(y)1/TAF9 mediates the transcriptional output of Notch signaling in Drosophila. J Cell Sci [Epub ahead of print]. PubMed ID: 25015288
Transcriptional activation of Notch signaling targets requires the formation of a ternary complex that involves the intracellular domain of the Notch receptor (NICD), DNA-binding protein Suppressor of Hairless [Su(H), RPBJ in mammals], and coactivator Mastermind (Mam). This study reports that E(y)1/TAF9, a component of the transcription factor TFIID complex, interacts specifically with the NICD/Su(H)/Mam complex to facilitate the transcriptional output of Notch signaling. E(y)1/TAF9 was identified in a large-scale in vivo RNAi screen for genes involved in a Notch-dependent mitotic-to-endocycle transition in Drosophila follicle cells. Knockdown of e(y)1/TAF9 displayed Notch-like phenotypes and defects in target gene and activity reporter expression in both the follicle cells and wing imaginal discs. Epistatic analyses in these two tissues indicate that E(y)1/TAF9 functions downstream of the Notch cleavage. Biochemical studies in S2 cells demonstrated that E(y)1/TAF9 physically interacts with the transcriptional effectors of Notch signaling, Su(H) and NICD. Together, these data suggest that the association of the NICD/Su(H)/Mastermind complex with E(y)1/TAF9 in response to Notch activation recruits the transcription initiation complex to induce Notch target genes, coupling Notch signaling with the transcriptional machinery.
Stork, T., Sheehan, A., Tasdemir-Yilmaz, O. E., Freeman, M. R. (2014). Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes. Neuron 83: 388-403. PubMed ID: 25033182
Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. This study characterized a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. The Fibroblast growth factor (FGF) receptor Heartless was demonstrated to autonomously control astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. It was further shown that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. The data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.
Pereira, A. M., Tudor, C., Pouille, P. A., Shekhar, S., Kanger, J. S., Subramaniam, V. and Martin-Blanco, E. (2014). Plasticity of the MAPK signaling network in response to mechanical stress. PLoS One 9: e101963. PubMed ID: 25025279
Cells display versatile responses to mechanical inputs and recent studies have identified the mitogen-activated protein kinase (MAPK) cascades mediating the biological effects observed upon mechanical stimulation. Although, MAPK pathways can act insulated from each other, several mechanisms facilitate the crosstalk between the components of these cascades. Yet, the combinatorial complexity of potential molecular interactions between these elements have prevented the understanding of their concerted functions. To analyze the plasticity of the MAPK signaling network in response to mechanical stress a non-saturating epistatic screen was performed in resting and stretched conditions employing as readout a JNK responsive dJun-FRET biosensor. By knocking down MAPKs, and JNK pathway regulators, singly or in pairs in Drosophila S2R+ cells, unexpected regulatory links were uncovered between JNK cascade kinases, Rho GTPases, MAPKs and the JNK phosphatase Puc. These relationships have been integrated in a system network model at equilibrium accounting for all experimentally validated interactions. This model allows predicting the global reaction of the network to its modulation in response to mechanical stress. It also highlights its context-dependent sensitivity.
Rahman, M., Nirala, N. K., Singh, A., Zhu, L. J., Taguchi, K., Bamba, T., Fukusaki, E., Shaw, L. M., Lambright, D. G., Acharya, J. K. and Acharya, U. R. (2014). Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity. J Cell Biol [Epub ahead of print]. PubMed ID: 25023514
Adenosine triphosphate (ATP) synthase β, the catalytic subunit of mitochondrial complex V, synthesizes ATP. This study shows that ATP synthase β is deacetylated by a human nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies display increased acetylation of specific Lys residues in ATP synthase β and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase β, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, this study unravel a ceramide-NAD+-sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD+ and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome.
Sunday, July 27th
Morais-de-Sa, E., Mukherjee, A., Lowe, N., St Johnston, D. (2014). Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity. Development 141: 2984-2992. PubMed ID: 25053432
The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. This study shows that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.
Skwarek, L. C., Windler, S. L., de Vreede, G., Rogers, G. C., Bilder, D. (2014). The F-box protein Slmb restricts the activity of aPKC to polarize epithelial cells. Development 141: 2978-2983. PubMed ID: 25053431
The Par-3/Par-6/aPKC complex is the primary determinant of apical polarity in epithelia across animal species, but how the activity of this complex is restricted to allow polarization of the basolateral domain is less well understood. In Drosophila, several multiprotein modules antagonize the Par complex through a variety of means. This study identified a new mechanism involving regulated protein degradation. Strong mutations in supernumerary limbs (slmb), which encodes the substrate adaptor of an SCF-class E3 ubiquitin ligase, cause dramatic loss of polarity in imaginal discs accompanied by tumorous proliferation defects. Slmb function is required to restrain apical aPKC activity in a manner that is independent of endolysosomal trafficking and parallel to the Scribble module of junctional scaffolding proteins. The involvement of the Slmb E3 ligase in epithelial polarity, specifically limiting Par complex activity to distinguish the basolateral domain, points to parallels with polarization of the C. elegans zygote.
Claret, S., Jouette, J., Benoit, B., Legent, K. and Guichet, A. (2014). PI(4,5)P2 produced by the PI4P5K SKTL controls apical size by tethering PAR-3 in Drosophila epithelial cells. Curr Biol 24: 1071-1079. PubMed ID: 24768049
The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides is controlled by the concerted action of phosphoinositide kinases and phosphatases. Using the Drosophila follicular epithelium as a model system in vivo, this study shows that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated by the PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, the disassembly was observed of adherens junctions, accompanied by actin cytoskeleton reorganization and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. Evidence is provided that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, PI(4,5)P2 was shown to be excluded from the cell apex, and PAR-3 was shown to diffuse laterally just prior to the apical constriction in a context of endogenous invagination. All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain.
Kupinski, A. P., Raabe, I., Michel, M., Ail, D., Brusch, L., Weidemann, T. and Bokel, C. (2013). Phosphorylation of the Smo tail is controlled by membrane localisation and is dispensable for clustering. J Cell Sci 126: 4684-4697. PubMed ID: 23943866
The Hedgehog (Hh) signalling cascade is highly conserved and involved in development and disease throughout evolution. Nevertheless, in comparison with other pathways, mechanistic understanding of Hh signal transduction is remarkably incomplete. In the absence of ligand, the Hh receptor Patched (Ptc) represses the key signal transducer Smoothened (Smo) through an unknown mechanism. Hh binding to Ptc alleviates this repression, causing Smo redistribution to the plasma membrane, phosphorylation and opening of the Smo cytoplasmic tail, and Smo oligomerisation. However, the order and interdependence of these events is as yet poorly understood. Smo activation was mathematically modelled and simulated for two alternative modes of pathway activation, with Ptc primarily affecting either Smo localisation or phosphorylation. Visualising Smo activation through a novel, fluorescence-based reporter allowed testing of these competing models. This study shows that Smo localisation to the plasma membrane is sufficient for phosphorylation of the cytoplasmic tail in the presence of Ptc. Using fluorescence cross-correlation spectroscopy, it was also demonstrated that inactivation of Ptc by Hh induces Smo clustering irrespective of Smo phosphorylation. These observations therefore support a model of Hh signal transduction whereby Smo subcellular localisation and not phosphorylation is the primary target of Ptc function.
Saturday, July 26th
Barrio, L., Dekanty, A. and Milan, M. (2014). MicroRNA-mediated regulation of Dp53 in the Drosophila fat body contributes to metabolic adaptation to nutrient deprivation. Cell Rep. PubMed ID: 25017064
Multiple conserved mechanisms sense nutritional conditions and coordinate metabolic changes in the whole organism. This study unravels a role for the Drosophila homolog of p53 (Dp53) in the fat body (FB; a functional analog of vertebrate adipose and hepatic tissues) in starvation adaptation. Under nutrient deprivation, FB-specific depletion of Dp53 accelerates consumption of major energy stores and reduces survival rates of adult flies. This study shows that Dp53 is regulated by the microRNA (miRNA) machinery and miR-305 in a nutrition-dependent manner. In well-fed animals, TOR signaling contributes to miR-305-mediated inhibition of Dp53. Nutrient deprivation reduces the levels of miRNA machinery components and leads to Dp53 derepression. These results uncover an organism-wide role for Dp53 in nutrient sensing and metabolic adaptation and open up avenues toward understanding the molecular mechanisms underlying p53 activation under nutrient deprivation.
van Mierlo, J. T., Overheul, G. J., Obadia, B., van Cleef, K. W., Webster, C. L., Saleh, M. C., Obbard, D. J. and van Rij, R. P. (2014). Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi. PLoS Pathog 10: e1004256. PubMed ID: 25032815
The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary 'arms race' may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. It has been shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. This study shows that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. Novel Nora-like viruses have been identified in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, it was found that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. These results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. It is suggested that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.
Ling, L., Ge, X., Li, Z., Zeng, B., Xu, J., Aslam, A. F., Song, Q., Shang, P., Huang, Y. and Tan, A. (2014). MicroRNA Let-7 regulates molting and metamorphosis in the silkworm, Bombyx mori. Insect Biochem Mol Biol [Epub ahead of print]. PubMed ID: 25016132
MicroRNAs (miRNAs) are a class of endogenous, non-coding, regulatory RNA molecules that post-transcriptionally regulate gene expression by binding to the 3'UTRs of mRNA targets and thus cause their degradation or translational inhibition. In insects, important roles of miRNAs in various biological processes have been demonstrated in Drosophila melanogaster. However, biological roles of miRNAs are barely unveiled in the majority of insect species due to limited genetic tools. This study introduces the transgenic miRNA sponge (miR-SP) technology combining with the binary GAL4/UAS system in the domesticated silkworm, Bombyx mori, to exploit the biological function of an evolutionally conserved miRNA, let-7 (see Drosophila let-7). Transgenic silkworm lines were successfully established in which a miRNA sponge construct targeting BmLet-7 seed region was expressed in a ubiquitous manner directed by A3-GAL4 driver. Transgenic animals showed decreased expression of BmLet-7, leading to developmental arrestment during the larval-larval and larval-pupal transition. Simultaneously, expression levels of the predicted BmLet-7 target genes, FTZ-F1 (see Drosophila Ftz-f1) and Eip74EF (E74; see Drosophila E74), key regulatory factors in the ecdysone pathway, were elevated in transgenic animals. The current study is the first report on application of the transgenic miR-SP technology in non-drosophilid insects, which will not only contribute to better understanding of let-7 biological roles, but also greatly facilitate future miRNA functional analysis in insects.
Rouhana, L., Weiss, J. A., King, R. S. and Newmark, P. A. (2014). PIWI homologs mediate Histone H4 mRNA localization to planarian chromatoid bodies. Development 141: 2592-2601. PubMed ID: 24903754
The well-known regenerative abilities of planarian flatworms are attributed to a population of adult stem cells called neoblasts that proliferate and differentiate to produce all cell types. A characteristic feature of neoblasts is the presence of large cytoplasmic ribonucleoprotein granules named chromatoid bodies, the function of which has remained largely elusive. This study shows that histone mRNAs are a common component of chromatoid bodies. The experiments also demonstrate that accumulation of histone mRNAs, which is typically restricted to the S phase of eukaryotic cells, is extended during the cell cycle of neoblasts. The planarian PIWI (see Drosophila Piwi) homologs SMEDWI-1 and SMEDWI-3 are required for proper localization of germinal histone H4 (gH4) mRNA to chromatoid bodies. The association between histone mRNA and chromatoid body components extends beyond gH4 mRNA, since transcripts of other core histone genes were also found in these structures. Additionally, piRNAs) corresponding to loci of every core histone type have been identified. Altogether, this work provides evidence that links PIWI proteins and chromatoid bodies to histone mRNA regulation in planarian stem cells. The molecular similarities between neoblasts and undifferentiated cells of other organisms raise the possibility that PIWI proteins might also regulate histone mRNAs in stem cells and germ cells of other metazoans.
Friday, July 25th
Olofsson, J., Sharp, K. A., Matis, M., Cho, B., Axelrod, J. D. (2014). Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity. Development 141: 2866-2874. PubMed ID: 25005476
Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, this study found that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification.
Ayukawa, T., Akiyama, M., Mummery-Widmer, J. L., Stoeger, T., Sasaki, J., Knoblich, J. A., Senoo, H., Sasaki, T., Yamazaki, M. (2014). Dachsous-Dependent Asymmetric Localization of Spiny-Legs Determines Planar Cell Polarity Orientation in Drosophila. Cell Rep [Epub ahead of print]. PubMed ID: 24998533
In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. This study shows that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. This model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.
Herranz, H., Weng, R. and Cohen, S. M. (2014). Crosstalk between Epithelial and Mesenchymal Tissues in Tumorigenesis and Imaginal Disc Development. Curr Biol 24: 1476-1484. PubMed ID: 24980505
Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. This study reports a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. Evidence that the secreted proteoglycan Perlecan can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. In conclusion, this study reports genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development.
Faisal, M. N., Hoffmann, J., El-Kholy, S., Kallsen, K., Wagner, C., Bruchhaus, I., Fink, C. and Roeder, T. (2014). Transcriptional regionalization of the fruit fly's airway epithelium. PLoS One 9: e102534. PubMed ID: 25020150
Although airway epithelia are primarily devoted to gas exchange, they have to fulfill a number of different tasks including organ maintenance and the epithelial immune response to fight airborne pathogens. These different tasks are at least partially accomplished by specialized cell types in the epithelium. In addition, a proximal to distal gradient mirroring the transition from airflow conduction to real gas exchange, is also operative. This study analyzed the trachea of larval Drosophila with respect to region-specific expression in the proximal to distal axis. The larval airway system is made of epithelial cells only. Differential expression was found between major trunks of the airways and more distal ones comprising primary, secondary and terminal ones. A more detailed analysis was performed using DNA-microarray analysis to identify cohorts of genes that are either predominantly expressed in the dorsal trunks or in the primary/secondary/terminal branches of the airways. Among these differentially expressed genes are especially those involved in signal transduction. Wnt-signalling associated genes for example are predominantly found in secondary/terminal airways. In addition, some G-protein coupled receptors are differentially expressed between both regions of the airways, exemplified by those activated by octopamine or tyramine, the invertebrate counterparts of epinephrine and norepinephrine. Whereas the OAMB is predominantly found in terminal airway regions, the octβ3R has higher expression levels in dorsal trunks. In addition, a significant association of genes regulated by hypoxia was observed with genes predominantly expressed in dorsal trunks; a similar level of association was observed between genes regulated by hypoxia and genes expressed in primary to terminal branches. Taken together, this observed differential expression is indicative for a proximal to distal transcriptional regionalization presumably reflecting functional differences in these parts of the fly's airway system.
Thursday, July 24th
Ehaideb, S. N., Iyengar, A., Ueda, A., Iacobucci, G. J., Cranston, C., Bassuk, A. G., Gubb, D., Axelrod, J. D., Gunawardena, S., Wu, C. F. and Manak, J. R. (2014). prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25024231
Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. This study shows that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. Enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, it was shown that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult Prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.
Moshfegh, Y., Bravo-Cordero, J. J., Miskolci, V., Condeelis, J. and Hodgson, L. (2014). A Trio-Rac1-Pak1 signalling axis drives invadopodia disassembly. Nat Cell Biol 16: 574-586. PubMed ID: 24859002
Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase (see Drosophila Rac) in invadopodia dynamics, a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor was developed for use in mammalian cell culture. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. This study describes an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.
Ellis, S. J., Goult, B. T., Fairchild, M. J., Harris, N. J., Long, J., Lobo, P., Czerniecki, S., Van Petegem, F., Schock, F., Peifer, M. and Tanentzapf, G. (2013). Talin autoinhibition is required for morphogenesis. Curr Biol 23: 1825-1833. PubMed ID: 24012314
The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that autoinhibits its activity. However, the functional consequence of such an interaction has not been explored in vivo. This study demonstrates that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, evidence is presented that talin autoinhibition is regulated by Rap1-dependent signaling. Based on these data, it is proposed that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis.
Bovellan, M., Romeo, Y., Biro, M., Boden, A., Chugh, P., Yonis, A., Vaghela, M., Fritzsche, M., Moulding, D., Thorogate, R., Jegou, A., Thrasher, A. J., Romet-Lemonne, G., Roux, P. P., Paluch, E. K. and Charras, G. (2014). Cellular Control of Cortical Actin Nucleation. Curr Biol [Epub ahead of print]. PubMed ID: 25017211
The contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division, migration, and tissue morphogenesis. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells and during division, where its misregulation can lead to aneuploidy. Despite its importance, knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence. This study used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 (see Drosophila Dia) and the Arp2/3 complex ( see Drosophila Arp2/3 component Actin-related protein 2/3 complex, subunit 1). Each nucleator contributes a similar amount of F-actin to the cortex but has very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. These findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator.
Wednesday, July 23rd
Bussell, J. J., Yapici, N., Zhang, S. X., Dickson, B. J., Vosshall, L. B. (2014). Abdominal-B Neurons Control Drosophila Virgin Female Receptivity. Curr Biol. PubMed ID: 24998527
Female sexual receptivity offers an excellent
model for complex behavioral decisions. The female must
parse her own reproductive state, the external environment,
and male sensory cues to decide whether to copulate. In
the fly Drosophila melanogaster, virgin female receptivity has
received relatively little attention, and its neural circuitry and
individual behavioral components remain unmapped. Using
a genome-wide neuronal RNAi screen, this study identified a subpopulation
of neurons responsible for pausing, a novel behavioral
aspect of virgin female receptivity characterized in this study. Abdominal-B (Abd-B), a homeobox transcription factor, was shown to be required in developing neurons for high
levels of virgin female receptivity. Silencing adult Abd-B neurons
significantly decreased receptivity. Two
components of receptivity were characterized that are elicited in sexually mature
females by male courtship: pausing and vaginal plate opening.
Silencing Abd-B neurons decreased pausing but did not affect
vaginal plate opening, demonstrating that these two components
of female sexual behavior are functionally separable. Synthetic
activation of Abd-B neurons increased pausing, but male
courtship song alone was not sufficient to elicit this behavior.
These results provide an entry point to the neural
circuit controlling virgin female receptivity. The female integrates
multiple sensory cues from the male to execute discrete
motor programs prior to copulation. Abd-B neurons control
pausing, a key aspect of female sexual receptivity, in response
to male courtship.
Hermann-Luibl, C., Yoshii, T., Senthilan, P. R., Dircksen, H. and Helfrich-Forster, C. (2014). The Ion Transport Peptide Is a New Functional Clock Neuropeptide in the Fruit Fly Drosophila melanogaster. J Neurosci 34: 9522-9536. PubMed ID: 25031396
The clock network of Drosophila melanogaster expresses various neuropeptides, but a function in clock-mediated behavioral control was so far only found for the neuropeptide Pigment dispersing factor (PDF). This paper proposes a role in the control of behavioral rhythms for the Ion transport peptide (ITP), which is expressed in the fifth small ventral lateral neuron, one dorsal lateral neuron, and in only a few nonclock cells in the brain. Immunocytochemical analyses revealed that ITP, like PDF, is most probably released in a rhythmic manner at projection terminals in the dorsal protocerebrum. This rhythm continues under constant dark conditions, indicating that ITP release is clock controlled. ITP expression is reduced in the hypomorph mutant Clk(AR), suggesting that ITP expression is regulated by CLOCK. Using a genetically encoded RNAi construct, ITP was knocked down in the two clock cells, and these flies were found to show reduced evening activity and increased nocturnal activity. Overexpression of ITP with two independent timeless-GAL4 lines completely disrupted behavioral rhythms, but only slightly dampened PER cycling in important pacemaker neurons, suggesting a role for ITP in clock output pathways rather than in the communication within the clock network. Simultaneous knockdown (KD) of ITP and PDF made the flies hyperactive and almost completely arrhythmic under constant conditions. Under light-dark conditions, the double-KD combined the behavioral characteristics of the single-KD flies. In addition, it reduced the flies' sleep. It is concluded that ITP and PDF are the clock's main output signals that cooperate in controlling the flies' activity rhythms.
Fedele, G., Green, E. W., Rosato, E. and Kyriacou, C. P. (2014). An electromagnetic field disrupts negative geotaxis in Drosophila via a CRY-dependent pathway. Nat Commun 5: 4391. PubMed ID: 25019586
Many higher animals have evolved the ability to use the Earth's magnetic field, particularly for orientation. Drosophila melanogaster also respond to electromagnetic fields (EMFs), although the reported effects are quite modest. This paper reports that negative geotaxis in flies, scored as climbing, is disrupted by a static EMF, and this is mediated by Cryptochrome (CRY), the blue-light circadian photoreceptor. CRYs may sense EMFs via formation of radical pairs of electrons requiring photoactivation of flavin adenine dinucleotide (FAD) bound near a triad of Trp residues, but mutation of the terminal Trp in the triad maintains EMF responsiveness in climbing. In contrast, deletion of the CRY C terminus disrupts EMF responses, indicating that it plays an important signalling role. CRY expression in a subset of clock neurons, or the photoreceptors, or the antennae, is sufficient to mediate negative geotaxis and EMF sensitivity. Climbing therefore provides a robust and reliable phenotype for studying EMF responses in Drosophila.
Chaves, I., van der Horst, G. T., Schellevis, R., Nijman, R. M., Koerkamp, M. G., Holstege, F. C., Smidt, M. P. and Hoekman, M. F. (2014). Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription. Curr Biol 24: 1248-1255. PubMed ID: 24856209
Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock, where it is integrated into the molecular oscillator via regulation of clock (see Drosophila Clock) gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals. As a consequence, peripheral oscillators can be uncoupled from the master clock when light and metabolic signals are not in phase. The signaling pathways responsible for coupling metabolic cues to the molecular clock are being rapidly uncovered. This study shows that insulin-phosphatidylinositol 3-kinase (PI3K)-Forkhead box class O3 (FOXO3 (see Drosophila Foxo)) signaling is required for circadian rhythmicity in the liver via regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, an effect that is rescued by overexpression of Clock. Subsequently, binding of FOXO3 to two Daf-binding elements (DBEs) located in the Clock promoter area was demonstrated, implicating Clock as a transcriptional target of FOXO3. Transcriptional oscillation of both core clock and output genes in the liver of FOXO3-deficient mice is affected, indicating a disrupted hepatic circadian rhythmicity. Finally, it was shown that insulin, a major regulator of FOXO activity, regulates Clock levels in a PI3K- and FOXO3-dependent manner. These data point to a key role of the insulin-FOXO3-Clock signaling pathway in the modulation of circadian rhythms.
Tuesday, July 21st
Rudolf, A., Buttgereit, D., Jacobs, M., Wolfstetter, G., Kesper, D., Putz, M., Berger, S., Renkawitz-Pohl, R., Holz, A. and Onel, S. F. (2014). Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion. BMC Cell Biol 15: 27. PubMed ID: 25000973
The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. Using fluorescence in situ hybridization and immunochemical analyses, an investigation was carried out of whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of WASp or its interaction partner wip had no defects in longitudinal fusion. These results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.
Hollfelder, D., Frasch, M. and Reim, I. (2014). Distinct functions of the laminin beta LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila. BMC Dev Biol 14: 26. PubMed ID: 24935095
The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos, multiple mutants were obtained for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin beta and collagen IV alpha1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV alpha2 chain is detected during late embryogenesis. It is concluded that assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. The data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the beta6/beta3/beta8 interface of the laminin beta LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.
Qing, Y., Yin, F., Wang, W., Zheng, Y., Guo, P., Schozer, F., Deng, H. and Pan, D. (2014). The Hippo effector Yorkie activates transcription by interacting with a histone methyltransferase complex through Ncoa6. Elife: e02564. PubMed ID: 25027438
The Hippo signaling pathway regulates tissue growth in Drosophila through the transcriptional coactivator Yorkie (Yki). How Yki activates target gene transcription is poorly understood. This study identified Nuclear receptor coactivator 6 (Ncoa6), a subunit of the Trithorax-related (Trr) histone H3 lysine 4 (H3K4) methyltransferase complex, as a Yki-binding protein. Like Yki, Ncoa6 and Trr are functionally required for Hippo-mediated growth control and target gene expression. Strikingly, artificial tethering of Ncoa6 to Sd is sufficient to promote tissue growth and Yki target expression even in the absence of Yki, underscoring the importance of Yki-mediated recruitment of Ncoa6 in transcriptional activation. Consistent with the established role for the Trr complex in histone methylation, this study showed that Yki, Ncoa6 and Trr are required for normal H3K4 methylation at Hippo target genes. These findings shed light on Yki-mediated transcriptional regulation and uncover a potential link between chromatin modification and tissue growth.
Robbins, R. M., Gbur, S. C. and Beitel, G. J. (2014). Non-Canonical Roles for Yorkie and Drosophila Inhibitor of Apoptosis 1 in Epithelial Tube Size Control. PLoS One 9: e101609. PubMed ID: 25036253
Precise control of epithelial tube size is critical for organ function, yet the molecular mechanisms remain poorly understood. This study examined the roles of cell growth and a highly conserved organ growth regulatory pathway in controlling the dimensions of the Drosophila tracheal (airway) system, a well-characterized system for investigating epithelial tube morphogenesis. Tracheal tube-size was found to be regulated in unexpected ways by the transcription factor Yorkie (Yki, homolog of mammalian YAP and TAZ) and the Salvador/Warts/Hippo (SWH) kinase pathway. Yki activity typically promotes cell division, inhibits apoptosis, and can promote cell growth. However, reducing Yki activity in developing embryos increases rather than decreases the length of the major tracheal tubes, the dorsal trunks (DTs). Similarly, reduction of Hippo pathway activity, which antagonizes Yki, shortens tracheal DTs. yki mutations do not alter DT cell volume or cell number, indicating that Yki and the Hippo pathway regulate cell shape and apical surface area, but not volume. Yki does not appear to act through known tracheal pathways of apical extracellular matrix, septate junctions (SJs), basolateral or tubular polarity. Instead, the Hippo pathway and Yki appear to act downstream or in parallel to SJs because a double mutant combination of an upstream Hippo pathway activator, kibra, and the SJ component sinu have the short tracheal phenotype of a kibra mutant. The critical target of Yki in tube size control is Drosophila Inhibitor of Apoptosis 1 (DIAP1), which in turn antagonizes the Drosophila effector caspase, Ice. Strikingly, there is no change in tracheal cell number in DIAP1 or Ice mutants, thus epithelial tube size regulation defines new non-apoptotic roles for Yki, DIAP1 and Ice.
Monday, July 21st
Huang, S., Zhang, Z., Zhang, C., Lv, X., Zheng, X., Chen, Z., Sun, L., Wang, H., Zhu, Y., Zhang, J., Yang, S., Lu, Y., Sun, Q., Tao, Y., Liu, F., Zhao, Y. and Chen, D. (2013). Activation of Smurf E3 ligase promoted by smoothened regulates hedgehog signaling through targeting patched turnover. PLoS Biol 11: e1001721. PubMed ID: 24302888
Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. This study identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, Smurf-mediated Patched ubiquitination was shown to depend on Smo activity in wing discs. Mechanistically, it was found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, the data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.
Fan, Y. and Bergmann, A. (2014). Multiple Mechanisms Modulate Distinct Cellular Susceptibilities toward Apoptosis in the Developing Drosophila Eye. Dev Cell [Epub ahead of print]. PubMed ID: 24981611
Although apoptosis is mechanistically well understood, a comprehensive understanding of how cells modulate their susceptibility toward apoptosis in a developing tissue is lacking. This study has revealed striking dynamics in the apoptotic susceptibilities of different cell types in the Drosophila retina over a period of only 24 hr. Mitotic cells are extremely susceptible to apoptotic signals, while postmitotic cells have developed several strategies to promote survival. For example, photoreceptor neurons accumulate the inhibitor of apoptosis, Diap1. In unspecified cells, Cullin-3-mediated degradation keeps Diap1 levels low. These cells depend on EGFR signaling for survival. As development proceeds, developmentally older photoreceptors degrade Diap1, resulting in increased apoptosis susceptibility. Finally, R8 photoreceptors have very efficient survival mechanisms independent of EGFR or Diap1. These examples illustrate how complex cellular susceptibility toward apoptosis is regulated in a developing organ. Similar complexities may regulate apoptosis susceptibilities in mammalian development, and tumor cells may take advantage of it.
Banerjee, S. and Chinthapalli, B. (2014). A proteomic screen with Drosophila Opa1-like identifies dMortalin as a regulator of mitochondrial morphology and cellular homeostasis. Int J Biochem Cell Biol. PubMed ID: 24998521
Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. This study shows Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with 'mitochondria-shaping' proteins like Mitochondrial assembly regulatory factor (Marf) and dMitofilin. To gain an insight into Opa1-like's network, this study delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (dMortalin) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of dmortalin in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in dmortalin RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing dmortalin RNAi. Taken together, this study has identified a novel interaction between dMortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.
Graybill, C. and Prehoda, K. E. (2014). Ordered multi-site phosphorylation of Lethal giant larvae by atypical Protein Kinase C. Biochemistry [Epub ahead of print]. PubMed ID: 25000553
In Par complex-mediated cell polarity, phosphorylation by atypical Protein Kinase C (aPKC) is coupled to substrate cortical displacement. Polarized substrates often contain multiple phosphorylation sites but the role of multi-site phosphorylation in Par-mediated polarity remains unclear. This study has dissected the role of the three aPKC phosphorylation sites within the tumor suppressor Lethal giant larvae (Lgl). Using a cultured Drosophila S2 cell cortical displacement assay, it was observed that phosphorylation at any one site only causes partial displacement. Complete displacement requires that all three sites are modified. A kinetic analysis was undertaken to determine if aPKC phosphorylates each site equivalently. As the sites are closely spaced, differences were not only observed in the rate of phosphorylation, but interaction between the sites. A complete description of the rates reveals a preferential order of phosphorylation. These results provide new insights to how multiple phosphorylation and phosphorylation rates could regulate localization behaviors of fate determinants at the cortex.
Sunday, June 20th
Maier, D., Cheng, S., Faubert, D. and Hipfner, D. R. (2014). A broadly conserved G-protein-coupled receptor kinase phosphorylation mechanism controls Drosophila Smoothened activity. PLoS Genet 10: e1004399. PubMed ID: 25009998
Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, four clusters of phosphorylation sites were mapped in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, it was found that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.
Ribeiro, P., Holder, M., Frith, D., Snijders, A. P. and Tapon, N. (2014). Crumbs promotes expanded recognition and degradation by the SCF(Slimb/beta-TrCP) ubiquitin ligase. Proc Natl Acad Sci U S A 111: E1980-1989. PubMed ID: 24778256
In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Slimb was identified as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function.
Bose, A., Majot, A. T. and Bidwai, A. P. (2014). The Ser/Thr Phosphatase PP2A Regulatory Subunit Widerborst Inhibits Notch Signaling. PLoS One 9: e101884. PubMed ID: 25006677
Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. wdb functions were also evaluated using mutations in Notch and E(spl) that affect the eye. It was found that the eye and R8 defects of the well-known Nspl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.
Santalla, M., Valverde, C. A., Harnichar, E., Lacunza, E., Aguilar-Fuentes, J., Mattiazzi, A. and Ferrero, P. (2014). Aging and CaMKII Alter Intracellular Ca2+ Transients and Heart Rhythm in Drosophila melanogaster. PLoS One 9: e101871. PubMed ID: 25003749
Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, this study reports changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduces spontaneous heart rate (HR) and increases the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.
Saturday, July 19th
Chen, G., Kong, J., Tucker-Burden, C., Anand, M., Rong, Y., Rahman, F., Moreno, C. S., Van Meir, E. G., Hadjipanayis, C. G. and Brat, D. J. (2014). Human Brat ortholog TRIM3 is a tumor suppressor that regulates asymmetric cell division in glioblastoma. Cancer Res. PubMed ID: 24947043
Cancer stem cells, capable of self-renewal and multipotent differentiation, influence tumor behavior through a complex balance of symmetric and asymmetric cell divisions. Mechanisms regulating the dynamics of stem cells and their progeny in human cancer are poorly understood. In Drosophila, mutation of brain tumor (brat) leads to loss of normal asymmetric cell division by developing neural cells and results in a massively enlarged brain composed of neuroblasts with neoplastic properties. Brat promotes asymmetric cell division and directs neural differentiation at least partially through its suppression on Myc. This study identified TRIM3 (11p15.5) as a human ortholog of Drosophila brat and demonstrates its regulation of asymmetric cell division and stem cell properties of glioblastoma (GBM), a highly malignant human brain tumor. TRIM3 gene expression is markedly reduced in human GBM samples, neurosphere cultures and cell lines and its reconstitution impairs growth properties in vitro and in vivo. TRIM3 expression attenuates stem-like qualities of primary GBM cultures, including neurosphere formation and the expression of stem cell markers CD133, Nestin and Nanog. In GBM stem cells, TRIM3 expression leads to a greater percentage dividing asymmetrically rather than symmetrically. As with Brat in Drosophila, TRIM3 suppresses c-Myc expression and activity in human glioma cell lines. A strong regulation of Musashi-Notch signaling by TRIM3 was demonstrated in GBM neurospheres and neural stem cells that may better explain its effect on stem cell dynamics. It is concludeed that TRIM3 acts as a tumor suppressor in GBM by restoring asymmetric cell division.
Xiao, G., Wan, Z., Fan, Q., Tang, X. and Zhou, B. (2014). The metal transporter ZIP13 supplies iron into the secretory pathway in Drosophila melanogaster. Elife: e03191. PubMed ID: 25006035
The intracellular iron transfer process is not well understood and the identity of the iron transporter responsible for iron delivery to the secretory compartments remains elusive. This study shows Drosophila ZIP13 (Slc39a13, CG7816 or Zip99C), a presumed zinc importer, fulfills the iron effluxing role. Interfering with dZIP13 expression causes iron-rescuable iron absorption defect, simultaneous iron increase in the cytosol and decrease in the secretory compartments, failure of ferritin iron loading, and abnormal collagen secretion. dZIP13 expression in E. coli confers upon the host iron-dependent growth and iron resistance. Importantly, time-coursed transport assays using an iron isotope indicated a potent iron exporting activity of dZIP13. The identification of dZIP13 as an iron transporter suggests that the spondylocheiro dysplastic form of Ehlers-Danlos syndrome, in which hZIP13 is defective, is likely due to a failure of iron delivery to the secretory compartments. These results also broaden knowledge of the scope of defects from iron dyshomeostasis.
Thackray, A. M., Di, Y., Zhang, C., Wolf, H., Pradl, L., Vorberg, I., Andreoletti, O. and Bujdoso, R. (2014). Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila. Biochem J. PubMed ID: 25000212
Prion diseases are fatal transmissible neurodegenerative diseases of various mammalian species. Central to these conditions is the conversion of the normal host protein PrPC into the abnormal conformer PrPSc. Mature PrPC is attached to the plasma membrane by a glycosylphosphatidylinositol anchor, while during biosynthesis and metabolism cytosolic, and secreted forms of the protein may arise. The role of topological PrPC variants in the mechanism of prion formation and prion-induced neurotoxicity during prion disease remains undefined. This study investigated whether Drosophila transgenic for ovine PrP targeted to the plasma membrane, to the cytosol, or for secretion, could produce transmissible toxicity following exposure to exogenous ovine prions. While all three topological variants of PrP were efficiently expressed in Drosophila, cytosolic PrP was conformationally distinct and required denaturation prior to recognition by immunobiochemical methods. Adult Drosophila transgenic for pan neuronally expressed ovine PrP targeted to the plasma membrane, to the cytosol, or for secretion, exhibited a decreased locomotor activity after exposure at the larval stage to ovine prions. Proteinase K-resistant PrPSc was detected by protein misfolding cyclic amplification in prion-exposed Drosophila transgenic for membrane-targeted PrP. Significantly, head homogenate from all three variants of prion-exposed PrP transgenic Drosophila induced a decreased locomotor activity when transmitted to PrP recipient flies. Drosophila transgenic for PrP targeted for secretion exhibited a spontaneous locomotor defect in the absence of prion exposure that was transmissible in PrP transgenic flies. These data are consistent with formation of transmissible prions in PrP transgenic Drosophila.
Das, S. S., Nanda, G. G. and Alone, D. P. (2014). Artemisinin and Curcumin inhibit Drosophila brain tumor, prolong life span, and restore locomotor activity. IUBMB Life. PubMed ID: 24975030
Deletion of tumor suppressor gene, lethal(2)giant larvae [l(2)gl], leads to brain tumor in Drosophila melanogaster at larval stage of development and severe brain dysplasia in mice. The effect of two potential antitumor drugs artemisinin and curcumin was studied in the perspective of inhibiting l(2)gl brain tumor. Efficacies of these drugs are characterized morphologically by measuring brain sizes of untreated and treated larvae on the basis of tumor inhibition and anatomically by looking at the cellular patterning via antibody staining of the third instar Drosophila larval brains. Behavioral experiments were done in form of locomotion to correlate tumor inhibition with the revival of brain function, and longevity assays were carried out to assess general health span. It was observed that both drugs show antitumor properties individually and in combination when larvae were treated with these drugs. Evidence was found for reactive oxygen species-mediated action of these drugs. Both the drugs when treated individually or together show better median life span and locomotory response. Although the efficacies of various treatments varied, overall, the positive effects of artemisinin and curcumin demonstrate a potential applicability of these drugs against brain tumor in higher organisms. It also paves a way for a simpler model system for screening such natural products for antitumor property.
Baxter, S. L., Allard, D. E., Crowl, C. and Sherwood, N. T. (2014). Cold temperature improves mobility and survival in drosophila models of Autosomal-Dominant Hereditary Spastic Paraplegia (AD-HSP). Dis Model Mech. PubMed ID: 24906373
Autosomal-Dominant Hereditary Spastic Paraplegia (AD-HSP) is a crippling neurodegenerative disease for which effective treatment or cure remains unknown. Victims experience progressive mobility loss due to degeneration of the longest axons in the spinal cord. Over half of AD-HSP cases arise from loss of function mutations in spastin, which encodes a microtubule-severing AAA ATPase. In Drosophila models of AD-HSP, larvae lacking Spastin exhibit abnormal motoneuron morphology and function, and most die as pupae. Adult survivors display impaired mobility, reminiscent of the human disease. This study shows that rearing pupae or adults at reduced temperature (18 °C), compared to the standard temperature of 24 °C, improves the survival and mobility of adult spastin mutants but leaves wild type flies unaffected. Flies expressing human spastin with pathogenic mutations are similarly rescued. Additionally, larval cooling partially rescues the larval synaptic phenotype. Cooling thus alleviates known spastin phenotypes for each developmental stage at which it is administered, and notably, is effective even in mature adults. It was further found that cold treatment rescues larval synaptic defects in mutants of Flower, a protein with no known relation to Spastin, and mobility defects in flies lacking Kat60-L1, another microtubule severing protein enriched in the CNS. Together, these data support that cold's beneficial effects extend beyond specific alleviation of Spastin dysfunction, to at least a subset of cellular and behavioral neuronal defects. Mild hypothermia, a common neuroprotective technique in clinical treatment of acute anoxia, may thus hold additional promise as a therapeutic approach for AD-HSP, and potentially, other neurodegenerative diseases.
Friday, July 18th
Akashi, M., Okamoto, A., Tsuchiya, Y., Todo, T., Nishida, E. and Node, K. (2014). A positive role for PERIOD in mammalian circadian gene expression. Cell Rep 7: 1056-1064. PubMed ID: 24794436
In the current model of the mammalian circadian clock, PERIOD (PER; see Drosophila Period) represses the activity of the circadian transcription factors BMAL1 (see Drosophila Cycle) and CLOCK (see Drosophila Clock), either independently or together with CRYPTOCHROME (CRY; see Drosophila Cry). This study provides evidence that PER has an entirely different function from that reported previously, namely, that PER inhibits CRY-mediated transcriptional repression through interference with CRY recruitment into the BMAL1-CLOCK complex. This indirect positive function of PER is consistent with previous data from genetic analyses using Per-deficient or mutant mice. Overall, the results support the hypothesis that PER plays different roles in different circadian phases: an early phase in which it suppresses CRY activity, and a later phase in which it acts as a transcriptional repressor with CRY. This buffering effect of PER on CRY might help to prolong the period of rhythmic gene expression. Additional studies are required to carefully examine the promoter- and phase-specific roles of PER.
Bedont, J. L., LeGates, T. A., Slat, E. A., Byerly, M. S., Wang, H., Hu, J., Rupp, A. C., Qian, J., Wong, G. W., Herzog, E. D., Hattar, S. and Blackshaw, S. (2014). Lhx1 controls terminal differentiation and circadian function of the suprachiasmatic nucleus. Cell Rep 7: 609-622. PubMed ID: 24767996
Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. This study shows that Lim homeodomain transcription factor 1 (Lhx1; see Drosophila Lim1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. These results identify a factor required for SCN terminal differentiation. In addition, this in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillator's role in physiology and cognition.
Malik, B. R. and Hodge, J. J. (2014). CASK and CaMKII function in Drosophila memory. Front Neurosci 8: 178. PubMed ID: 25009461
Calcium (Ca2+) and Calmodulin (CaM)-dependent serine/threonine kinase II (CaMKII) plays a central role in synaptic plasticity and memory due to its ability to phosphorylate itself and regulate its own kinase activity. Autophosphorylation at threonine 287 (T287) switches CaMKII to a Ca2+ independent and constitutively active state replicated by overexpression of a phosphomimetic CaMKII-T287D transgene or blocked by expression of a T287A transgene. A second pair of sites, T306 T307 in the CaM binding region once autophosphorylated, prevents CaM binding and inactivates the kinase during synaptic plasticity and memory, and can be blocked by a TT306/7AA transgene. Recently the synaptic scaffolding molecule called CASK (Ca2+/CaM-associated serine kinase) has been shown to control both sets of CaMKII autophosphorylation events during neuronal growth, Ca2+ signaling and memory in Drosophila. Deletion of either full length CASK or just its CaMK-like and L27 domains removed middle-term memory (MTM) and long-term memory (LTM), with CASK function in the α'/β' mushroom body neurons being required for memory. In a similar manner directly changing the levels of CaMKII autophosphorylation (T287D, T287A, or TT306/7AA) in the α'/β' neurons also removed MTM and LTM. In the CASK null mutant expression of either the Drosophila or human CASK transgene in the α'/β' neurons was found to completely rescue memory, confirming that CASK signaling in α'/β' neurons is necessary and sufficient for Drosophila memory formation and that the neuronal function of CASK is conserved between Drosophila and human. Expression of human CASK in Drosophila also rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation. Mutations in human CASK have recently been shown to result in intellectual disability and neurological defects suggesting a role in plasticity and learning possibly via regulation of CaMKII autophosphorylation.
Chen, X., Quan, Y., Wang, H. and Luo, H. (2014). Trehalase regulates neuroepithelial stem cell maintenance and differentiation in the Drosophila optic lobe. PLoS One 9: e101433. PubMed ID: 25003205
As one of the major hydrolases in Drosophila, Trehalase (Treh) catalyzes the hydrolysis of trehalose into glucose providing energy for flight muscle activity. Treh is highly conserved from bacteria to humans, but little is known about its function during animal development. This study analyzed the function of Treh in Drosophila optic lobe development. In the optic lobe, neuroepithelial cells (NEs) first divide symmetrically to expand the stem cell pool and then differentiate into neuroblasts, which divide asymmetrically to generate medulla neurons. This study found that the knockdown of Treh leads to a loss of the lamina and a smaller medulla. Analyses of Treh RNAi-expressing clones and loss-of-function mutants indicate that the lamina and medulla phenotypes result from neuroepithelial disintegration and premature differentiation into medulla neuroblasts. Although the principal role of Treh is to generate glucose, the Treh loss-of-function phenotype cannot be rescued by exogenous glucose. Thus, these results indicate that in addition to being a hydrolase, Treh plays a role in neuroepithelial stem cell maintenance and differentiation during Drosophila optic lobe development.
Thursday, July 17th
Stratoulias, V., Heino, T. I. and Michon, F. (2014). Lin-28 Regulates Oogenesis and Muscle Formation in Drosophila melanogaster. PLoS One 9: e101141. PubMed ID: 24963666
Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. This study reports critical roles for lin-28 during oogenesis. let-7 maturation was shown to be increased in lin-28 null mutant fly ovaries. lin-28 null mutant female flies display reduced fecundity, due to defects in egg chamber formation. More specifically, in mutant ovaries, the egg chambers were shown to fuse during early oogenesis resulting in abnormal late egg chambers. This phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. A model is suggested in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. These results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation.
Tripathy, R., Kunwar, P. S., Sano, H. and Renault, A. D. (2014). Transcriptional regulation of Drosophila gonad formation. Dev Biol [Epub ahead of print]. PubMed ID: 24927896
The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation a SGP cell autonomous role was identified for Midline (Mid) and Longitudinals lacking (Lola). These transcriptional factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms and an isoform specific requirement for lola was identified in the gonad that is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. These findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes.
Linnemannstons, K., Ripp, C., Honemann-Capito, M., Brechtel-Curth, K., Hedderich, M. and Wodarz, A. (2014). The PTK7-Related Transmembrane Proteins Off-track and Off-track 2 Are Co-receptors for Drosophila Wnt2 Required for Male Fertility. PLoS Genet 10: e1004443. PubMed ID: 25010066
Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. This study found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. An otk paralog (otk2), is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract (Linnemannstons, 2014).
Koride, S., He, L., Xiong, L. P., Lan, G., Montell, D. J. and Sun, S. X. (2014). Mechanochemical Regulation of Oscillatory Follicle Cell Dynamics in the Developing Drosophila Egg Chamber. Mol Biol Cell [Epub ahead of print]. PubMed ID: 24943847
During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by approximately 1.7 fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however the mechanisms generating the spatiotemporal pattern have been unclear. This study used a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. It is proposed that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho-kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. The model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, the prediction that removal of the basal lamina will increase the average oscillation period was tested. All together, the model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.
Wednesday, July 16th
Heimiller, J., Sridharan, V., Huntley, J., Wesley, C. S. and Singh, R. (2014). Drosophila Polypyrimidine Tract-Binding Protein (DmPTB) Regulates Dorso-Ventral Patterning Genes in Embryos. PLoS One 9: e98585. PubMed ID: 25014769
The Drosophila polypyrimidine tract-binding protein (dmPTB or Hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph03429, results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. This study used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5' and 3' splice site switching), alternative 5' ends, and mRNA level changes (up and down regulation). These findings are independently supported by RT-PCR analysis and in situ hybridization. A group of genes, such as zerknullt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral patterning and/or dorsal closure processes. Thus, loss of dmPTB function results in specific misregulated transcripts, including those that provide the missing link between the loss of dmPTB function and observed developmental defects in embryogenesis. This study provides the first comprehensive repertoire of genes affected in vivo in the heph mutant in Drosophila and offers insight into the role of dmPTB during embryonic development.
Davidovich, C., Goodrich, K. J., Gooding, A. R. and Cech, T. R. (2014). A dimeric state for PRC2. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 24992961
Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. This study shows that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression.
Ravens, S., Fournier, M., Ye, T., Stierle, M., Dembele, D., Chavant, V. and Tora, L. (2014). MOF-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation. Elife: e02104. PubMed ID: 24898753
The histone acetyltransferase (HAT) Mof is essential for mouse embryonic stem cells (mESC) pluripotency and early development. Mof is the enzymatic subunit of two different HAT complexes, male specific lethal (MSL) and non-specific lethal (NSL) (see Drosophila Dosage compensation). The individual contribution of MSL and NSL to transcription regulation in mESCs is not well understood. A genome-wide analysis show that 1) MSL and NSL bind to specific and common sets of expressed genes, 2) NSL binds exclusively at promoters, 3) while MSL binds in gene bodies. Nsl1 regulates proliferation and cellular homeostasis of mESCs. MSL is the main HAT acetylating H4K16 in mESCs, is enriched at many mESC-specific and bivalent genes. MSL is important to keep a subset of bivalent genes silent in mESCs, while developmental genes require MSL for expression during differentiation. Thus, NSL and MSL HAT complexes differentially regulate specific sets of expressed genes in mESCs and during differentiation.
Chelmicki, T., Dundar, F., Turley, M. J., Khanam, T., Aktas, T., Ramirez, F., Gendrel, A. V., Wright, P. R., Videm, P., Backofen, R., Heard, E., Manke, T. and Akhtar, A. (2014). MOF-associated complexes ensure stem cell identity and Xist repression. Elife 3: e02024. PubMed ID: 24842875
Histone acetyl transferases (HATs) play distinct roles in many cellular processes and are frequently misregulated in cancers. This study examined the regulatory potential of MYST1-(MOF)-containing MSL and NSL HAT complexes in mouse embryonic stem cells (ESCs) and neuronal progenitors. Both complexes influence transcription by targeting promoters and TSS-distal enhancers. In contrast to flies, the MSL complex is not exclusively enriched on the X chromosome, yet it is crucial for mammalian X chromosome regulation as it specifically regulates Tsix, the major repressor of Xist lncRNA. MSL depletion leads to decreased Tsix expression, reduced REX1 recruitment, and consequently, enhanced accumulation of Xist and variable numbers of inactivated X chromosomes during early differentiation. The NSL complex provides additional, Tsix-independent repression of Xist by maintaining pluripotency. MSL and NSL complexes therefore act synergistically by using distinct pathways to ensure a fail-safe mechanism for the repression of X inactivation in ESCs.
Volvert, M. L., Prevot, P. P., Close, P., Laguesse, S., Pirotte, S., Hemphill, J., Rogister, F., Kruzy, N., Sacheli, R., Moonen, G., Deiters, A., Merkenschlager, M., Chariot, A., Malgrange, B., Godin, J. D. and Nguyen, L. (2014). MicroRNA targeting of CoREST controls polarization of migrating cortical neurons. Cell Rep 7: 1168-1183. PubMed ID: 24794437
The migration of cortical projection neurons is a multistep process characterized by dynamic cell shape remodeling. The molecular basis of these changes remains elusive, and the present work describes how microRNAs (miRNAs) control neuronal polarization during radial migration. This study shows that miR-22 and miR-124 are expressed in the cortical wall where they target components of the CoREST/REST transcriptional repressor complex (see Drosophila CoRest), thereby regulating doublecortin transcription in migrating neurons. This molecular pathway underlies radial migration by promoting dynamic multipolar-bipolar cell conversion at early phases of migration, and later stabilization of cell polarity to support locomotion on radial glia fibers. Thus, this work emphasizes key roles of some miRNAs that control radial migration during cerebral corticogenesis.
Tuesday, July 15th
Alpatov, R., et al. (2014). A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response. Cell 157: 869-881. PubMed ID: 24813610
Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP (see Drosophila Fmr1). FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. This study identified FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, FMRP was shown to bind chromatin through its tandem Tudor (Agenet) domain in vitro, and it associates with chromatin in vivo. FMRP was also shown to participate in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.
Kahn, T. G., Stenberg, P., Pirrotta, V. and Schwartz, Y. B. (2014). Combinatorial Interactions Are Required for the Efficient Recruitment of Pho Repressive Complex (PhoRC) to Polycomb Response Elements. PLoS Genet 10: e1004495. PubMed ID: 25010632
Polycomb Group (PcG) proteins are epigenetic repressors that control metazoan development and cell differentiation. In Drosophila, PcG proteins form five distinct complexes targeted to genes by Polycomb Response Elements (PREs). Of all PcG complexes Pho Repressive Complex (PhoRC) is the only one that contains a sequence-specific DNA binding subunit (PHO or PHOL), which led to a model that places (PhoRC) at the base of the recruitment hierarchy. This study demonstrates that in vivo PHO is preferred to PHOL as a subunit of PhoRC and that PHO and PHOL associate with PREs and a subset of transcriptionally active promoters. Although the binding to the promoter sites depends on the quality of recognition sequences, the binding to PREs does not. Instead, the efficient recruitment of PhoRC to PREs requires the SFMBT subunit and crosstalk with Polycomb Repressive Complex 1. Human YY1 protein, the ortholog of PHO, was found to bind sites at active promoters in the human genome but does not bind most PcG target genes, presumably because the interactions involved in the targeting to Drosophila PREs are lost in the mammalian lineage. It is concluded that the recruitment of PhoRC to PREs is based on combinatorial interactions. Such a recruitment strategy is proposed to be important to attenuate the binding of PcG proteins when the target genes are transcriptionally active. These findings allow the appropriate placement of PhoRC in the PcG recruitment hierarchy and provide a rationale to explain why YY1 is unlikely to serve as a general recruiter of mammalian Polycomb complexes despite its reported ability to participate in PcG repression in flies.
Cooper, S., Dienstbier, M., Hassan, R., Schermelleh, L., Sharif, J., Blackledge, N. P., De Marco, V., Elderkin, S., Koseki, H., Klose, R., Heger, A. and Brockdorff, N. (2014). Targeting Polycomb to Pericentric Heterochromatin in Embryonic Stem Cells Reveals a Role for H2AK119u1 in PRC2 Recruitment. Cell Rep 7: 1456-1470. PubMed ID: 24857660
The mechanisms by which the major Polycomb group (PcG) complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, This study demonstrates that, in methylation-deficient embryonic stem cells (ESCs), CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 (see Drosophila H3) redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, it was found that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. These data are discussed in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications.
Van Bortle, K., Nichols, M. H., Li, L., Ong, C. T., Takenaka, N., Qin, Z. S. and Corces, V. G. (2014). Insulator function and topological domain border strength scale with architectural protein occupancy. Genome Biol 15: R82. PubMed ID: 24981874
Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and (see SMC-containing condensin complexes), an extensive pattern of colocalization was uncovered in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. This study has identified a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains.
Monday, July 14th
Hsu, Y. C., Li, L. and Fuchs, E. (2014). Transit-amplifying cells orchestrate stem cell activity and tissue regeneration. Cell 157: 935-949. PubMed ID: 24813615
Transit-amplifying cells (TACs) are an early intermediate in tissue regeneration. This study shows, using hair follicles (HFs) as a paradigm, that emerging TACs constitute a signaling center that orchestrates tissue growth. Whereas primed stem cells (SCs) generate TACs, quiescent SCs only proliferate after TACs form and begin expressing Sonic Hedgehog (SHH; see Drosophila Hedgehog). TAC generation is independent of autocrine SHH, but the TAC pool wanes if they can't produce SHH. This paradox is traced to two direct actions of SHH: promoting quiescent-SC proliferation and regulating dermal factors that stoke TAC expansion. Ingrained within quiescent SCs' special sensitivity to SHH signaling is their high expression of GAS1. Without sufficient input from quiescent SCs, replenishment of primed SCs for the next hair cycle is compromised, delaying regeneration and eventually leading to regeneration failure. These findings unveil TACs as transient but indispensable integrators of SC niche components and reveal an intriguing interdependency of primed and quiescent SC populations on tissue regeneration.
Papa, A., Wan, L., Bonora, M., Salmena, L., Song, M. S., Hobbs, R. M., Lunardi, A., Webster, K., Ng, C., Newton, R. H., Knoblauch, N., Guarnerio, J., Ito, K., Turka, L. A., Beck, A. H., Pinton, P., Bronson, R. T., Wei, W. and Pandolfi, P. P. (2014). Cancer-associated PTEN mutants act in a dominant-negative manner to suppress PTEN protein function. Cell 157: 595-610. PubMed ID: 24766807
PTEN (see Drosophila PTEN) dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. This study shows that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. Catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, Pten knockin mice were generated harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous PtenC124S/+ and PtenG129E/+ cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten+/- counterparts, whereas this difference is no longer apparent between PtenC124S/- and Pten-/- cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. These findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.
Ghosh, T., Aprea, J., Nardelli, J., Engel, H., Selinger, C., Mombereau, C., Lemonnier, T., Moutkine, I., Schwendimann, L., Dori, M., Irinopoulou, T., Henrion-Caude, A., Benecke, A. G., Arnold, S. J., Gressens, P., Calegari, F. and Groszer, M. (2014). MicroRNAs Establish Robustness and Adaptability of a Critical Gene Network to Regulate Progenitor Fate Decisions during Cortical Neurogenesis. Cell Rep 7: 1779-1788. PubMed ID: 24931612
Over the course of cortical neurogenesis, the transition of progenitors from proliferation to differentiation requires a precise regulation of involved gene networks under varying environmental conditions. In order to identify such regulatory mechanisms, this study analyzed microRNA (miRNA) target networks in progenitors during early and late stages of neurogenesis. Cyclin D1 (see Drosophila CyclinD) was found to be a network hub whose expression is miRNA-dosage sensitive. Experimental validation revealed a feedback regulation between cyclin D1 and its regulating miRNAs miR-20a, miR-20b, and miR-23a. Cyclin D1 induces expression of miR-20a and miR-20b, whereas it represses miR-23a. Inhibition of any of these miRNAs increases the developmental stage-specific mean and dynamic expression range (variance) of cyclin D1 protein in progenitors, leading to reduced neuronal differentiation. Thus, miRNAs establish robustness and stage-specific adaptability to a critical dosage-sensitive gene network during cortical neurogenesis. Understanding such network regulatory mechanisms for key developmental events can provide insights into individual susceptibilities for genetically complex neuropsychiatric disorders.
Yang, S. H., Kalkan, T., Morissroe, C., Marks, H., Stunnenberg, H., Smith, A. and Sharrocks, A. D. (2014). Otx2 and Oct4 Drive Early Enhancer Activation during Embryonic Stem Cell Transition from Naive Pluripotency. Cell Rep 7: 1968-1981. PubMed ID: 24931607
Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. A lot is known about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. This study examined the role of Otx2 (Drosophila homolog Orthodenticle) in this process in mouse ESCs and has demonstrated that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.
Sunday, July 13th
Rauskolb, C., Sun, S., Sun, G., Pan, Y. and Irvine, K. D. (2014). Cytoskeletal Tension Inhibits Hippo Signaling through an Ajuba-Warts Complex. Cell 158: 143-156. PubMed ID: 24995985
Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. This study reports that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein (jub), a negative regulator of Warts within the Hippo pathway. This study further shows that Jub associates with alpha-catenin and that its localization to adherens junctions and association with alpha-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. These observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth.
Azzolin, L., Panciera, T., Soligo, S., Enzo, E., Bicciato, S., Dupont, S., Bresolin, S., Frasson, C., Basso, G., Guzzardo, V., Fassina, A., Cordenonsi, M. and Piccolo, S. (2014). YAP/TAZ Incorporation in the beta-Catenin Destruction Complex Orchestrates the Wnt Response. Cell 158: 157-170. PubMed ID: 24976009
The Hippo transducers YAP/TAZ (see Drosophila Yorkie) have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. This study provides biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the beta-catenin (see Drosophila Armadillo) destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for beta-TrCP (see Drosophila Slimb) recruitment to the complex and beta-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/beta-catenin signaling. In line, the beta-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.
Dittmer, P. J., Dell'Acqua, M. L. and Sather, W. A. (2014). Ca(2+)/Calcineurin-Dependent Inactivation of Neuronal L-Type Ca(2+) Channels Requires Priming by AKAP-Anchored Protein Kinase A. Cell Rep 7: 1410-1416. PubMed ID: 24835998
Within neurons, Ca(2+)-dependent inactivation (CDI) of voltage-gated L-type Ca(2+) channels shapes cytoplasmic Ca(2+) signals. CDI is initiated by Ca(2+) binding to channel-associated calmodulin (see Drosophila Calmodulin) and subsequent Ca(2+)/calmodulin activation of the Ca(2+)-dependent phosphatase, calcineurin (CaN; see Drosophila Calcineurin), which is targeted to L channels by the A-kinase-anchoring protein AKAP79/150 (see Drosophila Akap200). This study reports that CDI of neuronal L channels is abolished by inhibition of PKA activity or PKA (see Drosophila PKA) anchoring to AKAP79/150 and that CDI is also suppressed by stimulation of PKA activity. Although CDI was reduced by positive or negative manipulation of PKA, interference with PKA anchoring or activity lowered Ca(2+) current density whereas stimulation of PKA activity elevated it. In contrast, inhibition of CaN reduced CDI but had no effect on current density. These results suggest a model wherein PKA-dependent phosphorylation enhances neuronal L current, thereby priming channels to undergo CDI, and Ca(2+)/calmodulin-activated CaN actuates CDI by reversing PKA-mediated enhancement of channel activity.
Wu, X. S., Zhang, Z., Zhao, W. D., Wang, D., Luo, F. and Wu, L. G. (2014). Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells. Cell Rep 7: 982-988. PubMed ID: 24835995
Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin (see Drosophila Calcineurin), which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), this study found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. It is therefore suggested that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.
Saturday, July 12th
Kohlmaier, A., Fassnacht, C., Jin, Y., Reuter, H., Begum, J., Dutta, D. and Edgar, B. A. (2014). Src kinase function controls progenitor cell pools during regeneration and tumor onset in the Drosophila intestine. Oncogene [Epub ahead of print]. PubMed ID: 24975577
Src non-receptor kinases have been implicated in events late in tumor progression. This paper reports a study of the role of Src kinases in the Drosophila intestinal stem cell (ISC) lineage, during tissue homeostasis and tumor onset. The adult Drosophila intestine contains only two progenitor cell types, division-capable ISCs and their daughters, postmitotic enteroblasts (EBs). Drosophila Src42a and Src64b were found to be required for optimal regenerative ISC division. Conversely, activation of Src42a, Src64b or another non-receptor kinase, Ack, promoted division of quiescent ISCs by coordinately stimulating G1/S and G2/M cell cycle phase progression. Prolonged Src kinase activation caused tissue overgrowth owing to cytokine receptor-independent Stat92E activation. This was not due to increased symmetric division of ISCs, but involved accumulation of weakly specified Notch+ but division-capable EB-like cells. Src activation triggered expression of a mitogenic module consisting of String/Cdc25 and Cyclin E that was sufficient to elicit division not only of ISCs but also of EBs. A small pool of similarly division-capable transit-amplifying Notch+ EBs was also identified in the wild type. Expansion of intermediate cell types that do not robustly manifest their transit-amplifying potential in the wild type may also contribute to regenerative growth and tumor development in other tissues in other organisms.
Lee, J. H., Bassel-Duby, R. and Olson, E. N. (2014). Heart- and muscle-derived signaling system dependent on MED13 and Wingless controls obesity in Drosophila. Proc Natl Acad Sci U S A 111: 9491-9496. PubMed ID: 24979807
Obesity develops in response to an imbalance of energy homeostasis and whole-body metabolism. Muscle plays a central role in the control of energy homeostasis through consumption of energy and signaling to adipose tissue. It has been reported previously that MED13, a subunit of the Mediator complex, acts in the heart to control obesity in mice. To further explore the generality and mechanistic basis of this observation, the potential influence of MED13 expression in heart and muscle on the susceptibility of Drosophila to obesity was investigated. Heart/muscle-specific knockdown of MED13 or MED12, another Mediator subunit, was shown to increase susceptibility to obesity in adult flies. To identify possible muscle-secreted obesity regulators, an RNAi-based genetic screen was performed of 150 genes that encode secreted proteins, and Wingless inhibition was shown to cause obesity. Consistent with these findings, muscle-specific inhibition of Armadillo, the downstream transcriptional effector of the Wingless pathway, also evoked an obese phenotype in flies. Epistasis experiments further demonstrated that Wingless functions downstream of MED13 within a muscle-regulatory pathway. Together, these findings reveal an intertissue signaling system in which Wingless acts as an effector of MED13 in heart and muscle and suggest that Wingless-mediated cross-talk between striated muscle and adipose tissue controls obesity in Drosophila. This signaling system appears to represent an ancestral mechanism for the control of systemic energy homeostasis.
Zhang, Q., Zhang, Y., Wu, L., Yang, Y., Li, X., Gao, L., Hou, X., Wu, Y., Hou, G., Li, Z. and Lin, X. (2014). dBrms1 Acts as a Positive Regulator of Notch Signaling in Drosophila Wing. J Genet Genomics 41: 317-325. PubMed ID: 24976121
The highly conserved Notch signaling is precisely regulated at different steps in a series of developmental events. However, little is known about the regulation of Notch receptor at transcriptional level. This study demonstrated that dBrms1 is involved in regulating Notch signaling in Drosophila wing. Knockdown of dBrms1 by RNA interference (RNAi) in wing disc suppresses the expression of Notch signaling target genes wingless (wg), cut and Enhancer of split m8 [E(spl)m8]. Consistently, the levels of Wg and Cut are reduced in the dBrms1 mutant clones. Importantly, loss of dBrms1 leads to significant reduction of Notch proteins. Furthermore, depletion of dBrms1 results in apparent downregulation of Notch transcription in the wing disc. Moreover, it was found that dBrms1 is functionally conserved with human Breast cancer metastasis suppressor 1 like (hBRMS1L) in the modulation of Notch signaling. Taken together, these data provide important insights into the biological function of dBrms1 in regulating Notch signaling.
Pekkurnaz, G., Trinidad, J. C., Wang, X., Kong, D. and Schwarz, T. L. (2014). Glucose Regulates Mitochondrial Motility via Milton Modification by O-GlcNAc Transferase. Cell 158: 54-68. PubMed ID: 24995978
Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. This study reports a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. The mitochondrial motor-adaptor protein Milton (also called TRAK1/2) was shown to be a required substrate for OGT to arrest mitochondrial motility, by mapping and mutating the key O-GlcNAcylated serine residues. The GlcNAcylation state of Milton is altered by extracellular glucose, and OGT was shown to alter mitochondrial motility in vivo. These findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.
Friday, July 11th
Miskiewicz, K., Jose, L. E., Yeshaw, W. M., Valadas, J. S., Swerts, J., Munck, S., Feiguin, F., Dermaut, B. and Verstreken, P. (2014). HDAC6 Is a Bruchpilot Deacetylase that Facilitates Neurotransmitter Release. Cell Rep [Epub ahead of print]. PubMed ID: 24981865
Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. This study shows that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (ALS). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. This work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission.
Chen, C. K., Bregere, C., Paluch, J., Lu, J. F., Dickman, D. K. and Chang, K. T. (2014). Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase. Nat Commun 5: 4246. PubMed ID: 24977345
Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signalling required for proper synaptic development, function and plasticity, particularly during changes in neuronal activity. This study presents evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). Mnb-dependent phosphorylation of Synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. These data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.
Cho, R. W., Kummel, D., Li, F., Baguley, S. W., Coleman, J., Rothman, J. E. and Littleton, J. T. (2014). Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24982161
Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca2+ influx while promoting evoked release in response to an action potential. Previous studies indicated Cpx may cross-link multiple SNARE complexes via a trans interaction to function as a fusion clamp. During Ca2+ influx, Cpx is predicted to undergo a conformational switch and collapse onto a single SNARE complex in a cis-binding mode to activate vesicle release. To test this model in vivo, structure-function studies of the Cpx protein was performed in Drosophila. Using genetic rescue approaches with cpx mutants that disrupt SNARE cross-linking, manipulations that are predicted to block formation of the trans SNARE array were found to disrupt the clamping function of Cpx. Unexpectedly, these same mutants rescue action potential-triggered release, indicating trans-SNARE cross-linking by Cpx is not a prerequisite for triggering evoked fusion. In contrast, mutations that impair Cpx-mediated cis-SNARE interactions that are necessary for transition from an open to closed conformation fail to rescue evoked release defects in cpx mutants, although they clamp spontaneous release normally. These in vivo genetic manipulations support several predictions made by the Cpx cross-linking model, but unexpected results suggest additional mechanisms are likely to exist that regulate Cpx's effects on SNARE-mediated fusion. These findings also indicate that the inhibitory and activating functions of Cpx are genetically separable, and can be mapped to distinct molecular mechanisms that differentially regulate the SNARE fusion machinery.
Li, Z., Johnson, M. R., Ke, Z., Chen, L. and Welte, M. A. (2014). Drosophila Lipid Droplets Buffer the H2Av Supply to Protect Early Embryonic Development. Curr Biol [Epub ahead of print]. PubMed ID: 24930966
Assembly of DNA into chromatin requires a delicate balancing act, as both dearth and excess of histones severely disrupt chromatin function. In particular, cells need to carefully control histone stoichiometry: if different types of histones are incorporated into chromatin in an imbalanced manner, it can lead to altered gene expression, mitotic errors, and death. Both the balance between individual core histones and the balance between core histones and histone variants are critical. This study finds that in early Drosophila embryos, histone balance in the nuclei is regulated by lipid droplets, cytoplasmic fat-storage organelles. Lipid droplets were previously known to function in long-term histone storage: newly laid embryos contain large amounts of excess histones generated during oogenesis, and the maternal supplies of core histone H2A and the histone variant H2Av are anchored to lipid droplets via the novel protein Jabba. In these embryos, synthesis of new H2A and H2Av is imbalanced, and newly produced H2Av can be recruited to lipid droplets. When droplet sequestration is disrupted by mutating Jabba, embryos display an elevated H2Av/H2A ratio in nuclei as well as mitotic defects, reduced viability, and hypersensitivity to H2Av overexpression. It is proposed that in Drosophila embryos, lipid droplets serve as a histone buffer, not only storing maternal histones to support the early cell cycles but also transiently sequestering H2Av produced in excess and thus ensuring proper histone balance in the nucleus.
Thursday, July 10th
Amcheslavsky, A., Nie, Y., Li, Q., He, F., Tsuda, L., Markstein, M. and Ip, Y. T. (2014). Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila. Development 141: 2621-2632. PubMed ID: 24961799
Intestinal stem cells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair. This study used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators. A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division defects, including loss of 5-ethynyl-2′-deoxyuridine (EdU) incorporation, phosphorylated histone 3 staining and expression of the mitotic protein Cdc2. Loss of Charlatan also led to a much reduced histone acetylation staining in precursor cells. Both the histone acetylation and ISC division defects could be rescued by the simultaneous decrease of the Histone Deacetylase 2. The overexpression of Charlatan blocked differentiation reversibly, but loss of Charlatan did not lead to automatic differentiation. The results together suggest that Charlatan does not simply act as an anti-differentiation factor but instead functions to maintain a chromatin structure that is compatible with stem cell properties, including proliferation.
You, J., Zhang, Y., Li, Z., Lou, Z., Jin, L. and Lin, X. (2014). Drosophila Perlecan Regulates Intestinal Stem Cell Activity via Cell-Matrix Attachment. Stem Cell Reports 2: 761-769. PubMed ID: 24936464
Stem cells require specialized local microenvironments, termed niches, for normal retention, proliferation, and multipotency. Niches are composed of cells together with their associated extracellular matrix (ECM). Currently, the roles of ECM in regulating niche functions are poorly understood. This study demonstrates that Perlecan (Pcan), a highly conserved ECM component, controls intestinal stem cell (ISC) activities and ISC-ECM attachment in Drosophila adult posterior midgut. Loss of Pcan from ISCs, but not other surrounding cells, causes ISCs to detach from underlying ECM, lose their identity, and fail to proliferate. These defects are not a result of a loss of epidermal growth factor receptor (EGFR) or JAK/STAT signaling activity but partially depend on integrin signaling activity. It is proposed that Pcan secreted by ISCs confers niche properties to the adjacent ECM that is required for ISC maintenance of stem cell identity, activity, and anchorage to the niche.
Park, J. S., Pyo, J. H., Na, H. J., Jeon, H. J., Kim, Y. S., Arking, R. and Yoo, M. A. (2014). Increased centrosome amplification in aged stem cells of the Drosophila midgut. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 24971546
Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Using anti-gamma-tubulin and anti-PH3 this study analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. These data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.
Rodgers, J. T., King, K. Y., Brett, J. O., Cromie, M. J., Charville, G. W., Maguire, K. K., Brunson, C., Mastey, N., Liu, L., Tsai, C. R., Goodell, M. A. and Rando, T. A. (2014). mTORC1 controls the adaptive transition of quiescent stem cells from G0 to GAlert. Nature 509: 393-396. PubMed ID: 24870234
A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function. The extent to which stem cells can regulate quiescence is unknown. This study shows that the stem cell quiescent state is composed of two distinct functional phases, G0 and an 'alert' phase termed GAlert. Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), this study shows that mTORC1 (see Drosophila Tor) activity is necessary and sufficient for the transition of satellite cells from G0 into GAlert and that signalling through the HGF receptor cMet is also necessary. G0-to-GAlert transitions were demonstrated in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. It is proposed that the transition of quiescent stem cells into GAlert functions as an 'alerting' mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.
Wednesday, July 9th
Yamulla, R. J., Kane, E. G., Moody, A. E., Politi, K. A., Lock, N. E., Foley, A. V. and Roberts, D. M. (2014). Testing Models of the APC Tumor Suppressor/β-catenin Interaction ReshapesαView of the Destruction Complex in Wnt Signaling. Genetics [Epub ahead of print]. PubMed ID: 24931405
The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor Adenomatous Polyposis Coli (APC) (see Drosophila APC) is an essential negative regulator of Wnt signaling inactivated in over 80% of colorectal cancers. APC participates in a multi-protein 'destruction complex' that targets the proto-oncogene β-catenin (see Drosophila Armadillo) for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high affinity β-catenin binding-sites (20 amino acid repeats; 20Rs) on APC. This study tested these models by generating a Drosophila APC2 mutant lacking all β-catenin binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. The results are inconsistent with current models, as it was found that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, an APC2 mutant was generated lacking all β-catenin binding-sites (including the 15Rs), and a direct β-catenin/APC interaction was found to be also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, these findings support a model whereby β-catenin binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes a separation of β-catenin destruction and Wg/Wnt signaling outputs was observed, and it is suggested that cytoplasmic retention of β-catenin likely accounts for this difference.
Mao, F., Yang, X., Fu, L., Lv, X., Zhang, Z., Wu, W., Yang, S., Zhou, Z., Zhang, L. and Zhao, Y. (2014). Kto-Skd complex can regulate ptc expression by interacting with Ci in Hedgehog signaling pathway. J Biol Chem [Epub ahead of print]. PubMed ID: 24962581
Hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments which derive from adjacent cell populations. The anterior/posterior (A/P) boundaries which are critical to maintain the position of organizers are established by a complex mechanism involving the Hh signaling. This study uncovered the regulation of ptc in Hh signaling pathway which is contributive to A/P boundary formation by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, further evidence is provided that Kto-Skd affects the A/P-axial development of the whole wing disc. And Kto can interact with Cubitus interruptus (Ci) and bind to the Ci-binding region on the ptc promoter. Both subunits are regulated by Hh signals to downregulate ptc expression.
Gleixner, E. M., Canaud, G., Hermle, T., Guida, M. C., Kretz, O., Helmstadter, M., Huber, T. B., Eimer, S., Terzi, F. and Simons, M. (2014). V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis. Cell Rep [Epub ahead of print]. PubMed ID: 24953654
mTOR kinase (see Drosophila Target of rapamycin) is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, this study showa that the V-ATPase is required for Rheb-dependent epithelial growth. A positive feedback loop for the control of apical protein uptake was uncovered that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, evidence is provided that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, these findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions.
Lee, Y. S., Kim, J. W., Osborne, O., Oh da, Y., Sasik, R., Schenk, S., Chen, A., Chung, H., Murphy, A., Watkins, S. M., Quehenberger, O., Johnson, R. S. and Olefsky, J. M. (2014). Increased Adipocyte O2 Consumption Triggers HIF-1alpha, Causing Inflammation and Insulin Resistance in Obesity. Cell 157: 1339-1352. PubMed ID: 24906151
Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. This study reports that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1alpha induction (see Drosophila similar), setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1alpha can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance.
Lu, M., Zak, J., Chen, S., Sanchez-Pulido, L., Severson, D. T., Endicott, J., Ponting, C. P., Schofield, C. J. and Lu, X. (2014). A code for RanGDP binding in ankyrin repeats defines a nuclear import pathway. Cell 157: 1130-1145. PubMed ID: 24855949
Regulation of nuclear import is fundamental to eukaryotic biology. The majority of nuclear import pathways are mediated by importin-cargo interactions. Yet not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. This study identified a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP(see Drosophila Ran) efficiently, and consequently enter the nucleus. This code, experimentally tested in 17 ARPs, predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy and is acquired by the most common familial melanoma-associated CDKN2A mutation, leading to nuclear accumulation of mutant p16ink4a. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway and is frequently used by AR-containing transcriptional regulators, especially those regulating NF-kappaB/p53.
Tuesday, July 8th
Weidmann, C. A., Raynard, N. A., Blewett, N. H., Van Etten, J. and Goldstrohm, A. C. (2014). The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation. RNA [Epub ahead of print]. PubMed ID: 24942623
PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. This study investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. A previously proposed model was evaluated wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, it was found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, it was determined that the poly(A) is necessary for repression by the RBD. These results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, it was shown that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, these data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation.
Ghosh, S., Obrdlik, A., Marchand, V. and Ephrussi, A. (2014). The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila. PLoS Genet 10: e1004455. PubMed ID: 24967911
In eukaryotes, RNA processing events in the nucleus influence the fate of transcripts in the cytoplasm. The multi-protein exon junction complex (EJC) associates with mRNAs concomitant with splicing in the nucleus and plays important roles in export, translation, surveillance and localization of mRNAs in the cytoplasm. In mammalian cells, the ribosome associated protein PYM (HsPYM) binds the Y14-Mago heterodimer moiety of the EJC core, and disassembles EJCs, presumably during the pioneer round of translation. However, the significance of the association of the EJC with mRNAs in a physiological context has not been tested and the function of PYM in vivo remains unknown. This study addresses PYM function in Drosophila, where the EJC core proteins are genetically required for oskar mRNA localization during oogenesis. Evidence is provided that the EJC binds oskar mRNA in vivo. Using an in vivo transgenic approach, it was shown that elevated amounts of the Drosophila PYM (DmPYM) N-terminus during oogenesis cause dissociation of EJCs from oskar RNA, resulting in its mislocalization and consequent female sterility. In contrast to HsPYM, DmPYM does not interact with the small ribosomal subunit and dismantles EJCs in a translation-independent manner upon over-expression. Biochemical analysis shows that formation of the PYM-Y14-Mago ternary complex is modulated by the PYM C-terminus revealing that DmPYM function is regulated in vivo. Furthermore, it was found that whereas under normal conditions DmPYM is dispensable, its loss of function is lethal to flies with reduced y14 or mago gene dosage. This analysis demonstrates that the amount of DmPYM relative to the EJC proteins is critical for viability and fertility. This, together with the fact that the EJC-disassembly activity of DmPYM is regulated, implicates PYM as an effector of EJC homeostasis in vivo.
Mulvey, B. B., Olcese, U., Cabrera, J. R. and Horabin, J. I. (2014). An interactive network of long non-coding RNAs facilitates the Drosophila sex determination decision. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 24954180
Genome analysis in several eukaryotes shows a surprising number of transcripts which do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. This study found that Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, SxlPe, which must be activated to specify female sex. They are primarily from two regions, R1 and R2, upstream of SxlPe and show a dynamic developmental profile. Of the four lncRNA strands only one, R2 antisense, has its peak coincident with SxlPe transcription, suggesting that it may promote activation. Indeed, its expression is regulated by the X chromosome counting genes, whose dose determines whether SxlPe is transcribed. Transgenic lines which ectopically express each of the lncRNAs show they can act in trans, not only impacting the process of sex determination but also altering the levels of the other lncRNAs. Generally, expression of R1 is negative whereas R2 is positive to females. This ectopic expression also results in a change in the local chromatin marks, affecting the timing and strength of SxlPe transcription. The chromatin marks are those deposited by the Polycomb and Trithorax groups of chromatin modifying proteins, which were found bind to the lncRNAs. It is suggested that the increasing numbers of non-coding transcripts being identified are a harbinger of interacting networks similar to the one this study describes.
Murota, Y., Ishizu, H., Nakagawa, S., Iwasaki, Y. W., Shibata, S., Kamatani, M. K., Saito, K., Okano, H., Siomi, H. and Siomi, M. C. (2014). Yb Integrates piRNA Intermediates and Processing Factors into Perinuclear Bodies to Enhance piRISC Assembly. Cell Rep [Epub ahead of print]. PubMed ID: 24953657
PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. This study shows that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. It is suggested that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.
Monday, July 7th
Cho, A., Tang, Y., Davila, J., Deng, S., Chen, L., Miller, E., Wernig, M. and Graef, I. A. (2014). Calcineurin signaling regulates neural induction through antagonizing the BMP pathway. Neuron 82: 109-124. PubMed ID: 24698271
Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing mammalian embryos. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. This study found that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins (see Drosophila Mad). Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and the development of anterior structures.
Xu, K., Wu, Z., Renier, N., Antipenko, A., Tzvetkova-Robev, D., Xu, Y., Minchenko, M., Nardi-Dei, V., Rajashankar, K. R., Himanen, J., Tessier-Lavigne, M. and Nikolov, D. B. (2014). Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism. Science 344: 1275-1279. PubMed ID: 24876346
Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC; Drosophila homolog Frazzled) is a well-established netrin-1 receptor mediating attractive responses. Evidence is provided that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. This study determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.
Schmalen, I., Reischl, S., Wallach, T., Klemz, R., Grudziecki, A., Prabu, J. R., Benda, C., Kramer, A. and Wolf, E. (2014). Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. Cell 157: 1203-1215. PubMed ID: 24855952
Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. The structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. Evidence is provided that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. These studies may allow for the development of circadian and metabolic modulators.
Seo, J., Giusti-Rodriguez, P., Zhou, Y., Rudenko, A., Cho, S., Ota, K. T., Park, C., Patzke, H., Madabhushi, R., Pan, L., Mungenast, A. E., Guan, J. S., Delalle, I. and Tsai, L. H. (2014). Activity-dependent p25 generation regulates synaptic plasticity and Abeta-induced cognitive impairment. Cell 157: 486-498. PubMed ID: 24725413
Cyclin-dependent kinase 5 (see Drosophila Cdk5) regulates numerous neuronal functions with its activator, p35. Under neurotoxic conditions, p35 undergoes proteolytic cleavage to liberate p25, which has been implicated in various neurodegenerative diseases. This study shows that p25 is generated following neuronal activity under physiological conditions in a GluN2B- and CaMKIIalpha-dependent manner (GluN2B is a subunit of the NMDA receptor and see Drosophila CaMKII). Moreover, a knockin mouse model was developed in which endogenous p35 is replaced with a calpain-resistant mutant p35 (Deltap35KI) to prevent p25 generation. The Deltap35KI mice exhibit impaired long-term depression and defective memory extinction, likely mediated through persistent GluA1 (see Drosophila Glu-RIIA and Glu-RIIB) phosphorylation at Ser845. Finally, crossing the Deltap35KI mice with the 5XFAD mouse model of Alzheimer's disease (AD) resulted in an amelioration of β-amyloid (Abeta)-induced synaptic depression and cognitive impairment. Together, these results reveal a physiological role of p25 production in synaptic plasticity and memory and provide new insights into the function of p25 in Abeta-associated neurotoxicity and AD-like pathology.
Sunday, June 6th
Abouchar, L., Petkova, M. D., Steinhardt, C. R. and Gregor, T. (2014). Fly wing vein patterns have spatial reproducibility of a single cell. J R Soc Interface 11. PubMed ID: 24942847
Developmental processes in multicellular organisms occur in fluctuating environments and are prone to noise, yet they produce complex patterns with astonishing reproducibility. This study measured the left-right and inter-individual precision of bilaterally symmetric fly wings across the natural range of genetic and environmental conditions and found that wing vein patterns are specified with identical spatial precision and are reproducible to within a single-cell width. The early fly embryo operates at a similar degree of reproducibility, suggesting that the overall spatial precision of morphogenesis in Drosophila performs at the single-cell level. Could development be operating at the physical limit of what a biological system can achieve?
Oliveira, M. M., Shingleton, A. W. and Mirth, C. K. (2014). Coordination of Wing and Whole-Body Development at Developmental Milestones Ensures Robustness against Environmental and Physiological Perturbations. PLoS Genet 10: e1004408. PubMed ID: 24945255
Development produces correctly patterned tissues under a wide range of conditions that alter the rate of development in the whole body. Two hypotheses are proposed through which tissue patterning could be coordinated with whole-body development to generate this robustness. The first hypothesis states that tissue patterning is tightly coordinated with whole-body development over time. The second hypothesis is that tissue patterning aligns at developmental milestones. To distinguish between these two hypotheses, a staging scheme for the wing imaginal discs of Drosophila larvae was developed using the expression of canonical patterning genes, linking this scheme to three whole-body developmental events: moulting, larval wandering and pupariation. This scheme was used to explore how the progression of pattern changes when developmental time is altered either by changing temperature or by altering the timing of hormone synthesis that drives developmental progression. It was found the expression pattern in the wing disc always aligned at moulting and pupariation, indicating that these key developmental events represent milestones. Between these milestones, the progression of pattern showed greater variability in response to changes in temperature and alterations in physiology. Furthermore, the data showed that discs from wandering larvae showed greater variability in patterning stage. Thus for wing disc patterning, wandering does not appear to be a developmental milestone. These findings reveal that tissue patterning remains robust against environmental and physiological perturbations by aligning at developmental milestones. Furthermore, the work provides an important glimpse into how the development of individual tissues is coordinated with the body as a whole.
Chen, D., Qu, C. and Hewes, R. S. (2014). Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 24931409
Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, a screen was conducted of 545 splice-trap lines and 28 loci were identified that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and Shep proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple pre-synaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, though not entirely, normal locomotor behavior. Thus, shep plays an important role in the metamorphic development of many neurons.
Lopez-Rios, J., et al. (2014). Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature 511: 46–51. PubMed ID: 24990743
The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. This study analysed bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene (see Drosophila Ptc), which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. This study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.
Saturday, July 5th
Ma, H., Xu, H. and O'Farrell, P. H. (2014). Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster. Nat Genet 46: 393-397. PubMed ID: 24614071
It is not known how selection affects mutations in the multiple copies of the mitochondrial genome. This study transferred cytoplasm between D. melanogaster embryos carrying mitochondrial mutations to create heteroplasmic lines transmitting two mitochondrial genotypes. Increased temperature imposed selection against a temperature-sensitive mutation affecting cytochrome oxidase, driving decreases in the abundance of the mutant genome over successive generations. Selection did not influence the health or fertility of the flies but acted during midoogenesis to influence competition between the genomes. Mitochondria might incur an advantage through selective localization, survival or proliferation, yet timing and insensitivity to park mutation suggest that preferential proliferation underlies selection. Selection drove complete replacement of the temperature-sensitive mitochondrial genome by a wild-type genome but also stabilized the multigenerational transmission of two genomes carrying complementing detrimental mutations. While they are so balanced, these stably transmitted mutations have no detrimental phenotype, but their segregation could contribute to disease phenotypes and somatic aging.
Jung, H., Mazzoni, E. O., Soshnikova, N., Hanley, O., Venkatesh, B., Duboule, D. and Dasen, J. S. (2014). Evolving hox activity profiles govern diversity in locomotor systems. Dev Cell 29: 171-187. PubMed ID: 24746670
The emergence of limb-driven locomotor behaviors was a key event in the evolution of vertebrates and fostered the transition from aquatic to terrestrial life. This study showed that the generation of limb-projecting lateral motor column (LMC) neurons in mice relies on a transcriptional autoregulatory module initiated via transient activity of multiple genes within the HoxA and HoxC clusters. Repression of this module at thoracic levels restricts expression of LMC determinants, thus dictating LMC position relative to the limbs. This suppression is mediated by a key regulatory domain that is specifically found in the Hoxc9 proteins (Drosophila homolog: AbdB) of appendage-bearing vertebrates. The profile of Hoxc9 expression inversely correlates with LMC position in land vertebrates and likely accounts for the absence of LMC neurons in limbless species such as snakes. Thus, modulation of both Hoxc9 protein function and Hoxc9 gene expression likely contributed to evolutionary transitions between undulatory and ambulatory motor circuit connectivity programs.
Cressy, M., Valente, D., Altick, A., Kockenmesiter, E., Honegger, K., Qin, H., Mitra, P. P. and Dubnau, J. (2014). Laboratory evolution of adenylyl cyclase independent learning in Drosophila and missing heritability. Genes Brain Behav 13(6) :565-77. PubMed ID: 24888634
Gene interactions are acknowledged to be a likely source of missing heritability in large-scale genetic studies of complex neurological phenotypes. However, involvement of rare variants, de novo mutations, genetic lesions that are not easily detected with commonly used methods and epigenetic factors also are possible explanations. This study used a laboratory evolution study to investigate the modulatory effects of background genetic variation on the phenotypic effect size of a null mutation with known impact on olfactory learning. To accomplish this, a population was first established that contained variation at just 23 loci, and selection was used to evolve suppression of the learning defect seen with null mutations in the rutabaga adenylyl cyclase. Thus the system was biased to favor relatively simplified outcomes by choosing a Mendelian trait and by restricting the genetic variation segregating in the population. This experimental design also assures that the causal effects are among the known 23 segregating loci. A robust response was observed to selection that requires the presence of the 23 variants. Analyses of the underlying genotypes showed that interactions between more than two loci are likely to be involved in explaining the selection response, with implications for the missing heritability problem.
Ben-Johny, M., Yang, P. S., Niu, J., Yang, W., Joshi-Mukherjee, R. and Yue, D. T. (2014). Conservation of Ca(2+)/Calmodulin Regulation across Na and Ca(2+) Channels. Cell 157: 1657-1670. PubMed ID: 24949975
Voltage-gated Na and Ca(2+) channels comprise distinct ion channel superfamilies, yet the carboxy tails of these channels exhibit high homology, hinting at a long-shared and purposeful module. For different Ca(2+) channels, carboxyl-tail interactions with calmodulin (see Drosophila Cam) do elaborate robust and similar forms of Ca(2+) regulation. However, Na channels have only shown subtler Ca(2+) modulation that differs among reports, challenging attempts at unified understanding. In this study, by rapid Ca(2+) photorelease onto Na channels, this view of Na channel regulation has been reset. For cardiac-muscle channels (NaV1.5), reported effects from which most mechanistic proposals derive, no Ca(2+) modulation was observed. Conversely, for skeletal-muscle channels (NaV1.4), fast Ca(2+) regulation was uncovered eerily similar to that of Ca(2+) channels. Channelopathic myotonia mutations halve NaV1.4 Ca(2+) regulation, and transplanting the NaV1.4 carboxy tail onto Ca(2+) channels recapitulates Ca(2+) regulation. Thus, this study argues for the persistence and physiological relevance of an ancient Ca(2+) regulatory module across Na and Ca(2+) channels.
Friday, July 4th
Li, Q., Lewandowski, J. P., Powell, M. B., Norrie, J. L., Cho, S. H. and Vokes, S. A. (2014). A Gli silencer is required for robust repression of gremlin in the vertebrate limb bud. Development 141: 1906-1914. PubMed ID: 24700818
The transcriptional response to the Hedgehog (Hh) pathway is mediated by Gli proteins (see Drosophila Cubitus interruptus), which function as context-dependent transcriptional activators or repressors. However, the mechanism by which Gli proteins regulate their target genes is poorly understood. This is the first genetic characterization of a Gli-dependent cis-regulatory module (CRM), focusing on its regulation of Grem1 in the mouse limb bud. The CRM, termed GRE1 (Gli responsive element 1), can act as both an enhancer and a silencer. The enhancer activity requires sustained Hh signaling. As a Gli-dependent silencer, GRE1 prevents ectopic transcription of Grem1 driven through additional CRMs. In doing so, GRE1 works with additional GREs to robustly regulate Grem1. It is suggested that multiple Gli CRMs may be a general mechanism for mediating a robust transcriptional response to the Hh pathway.
Groschel, S., et al. (2014). A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia. Cell 157: 369-381. PubMed ID: 24703711
Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1 (Drosophila homolog: Hamlet). Applying functional genomics and genome-engineering, this study demonstrated that both 3q rearrangements reposition a distal GATA2 enhancer (see Drosophila Serpent) to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. These data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.
Pearson, J. C. and Crews, S. T. (2014). Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression. Dev Biol [Epub ahead of print]. PubMed ID: 24854999
Transcriptional enhancers integrate information derived from transcription factor binding to control gene expression. One key question concerns the extent of trans- and cis-regulatory variation in how co-expressed genes are controlled. The Drosophila CNS midline cells constitute a group of neurons and glia in which expression changes can be readily characterized during specification and differentiation. Using a transgenic approach, the cis-regulation of multiple genes expressed in the Drosophila CNS midline primordium cells were compared, and it was shown that while the expression patterns may appear alike, the target genes are not equivalent in how these common expression patterns are achieved. Some genes utilize a single enhancer that promotes expression in all midline cells, while others utilize multiple enhancers with distinct spatial, temporal, and quantitative contributions. Two regulators, Single-minded and Notch, play key roles in controlling early midline gene expression. While Single-minded is expected to control expression of most, if not all, midline primordium-expressed genes, the role of Notch in directly controlling midline transcription is unknown. Midline primordium expression of the rhomboid gene is dependent on cell signaling by the Notch signaling pathway. Mutational analysis of a rhomboid enhancer reveals at least 5 distinct types of functional cis-control elements, including a binding site for the Notch effector, Suppressor of Hairless. The results suggest a model in which Notch/Suppressor of Hairless levels are insufficient to activate rhomboid expression by itself, but does so in conjunction with additional factors, some of which, including Single-minded, provide midline specificity to Notch activation. Similarly, a midline glial enhancer from the argos gene, which is dependent on EGF/Spitz signaling, is directly regulated by contributions from both Pointed, the EGF transcriptional effector, and Single-minded. In contrast, midline primordium expression of other genes shows a strong dependence on Single-minded and varying combinations of additional transcription factors. Thus, Single-minded directly regulates midline primordium-expressed genes, but in some cases plays a primary role in directing target gene midline expression, and in others provides midline specificity to cell signaling inputs.
Ordway, A., Hancuch, K. N., Johnson, W., Wiliams, T. M. and Rebeiz, M. (2014). The expansion of body coloration involves coordinated evolution in cis and trans within the pigmentation regulatory network of Drosophila prostipennis. Dev Biol [Epub ahead of print]. PubMed ID: 24907418
The generation of complex morphological features requires the precisely orchestrated expression of numerous genes during development. While several traits have been resolved to evolutionary changes within a single gene, the evolutionary path by which genes derive co-localized or mutually excluded expression patterns is currently a mystery. This study investigated how the Drosophila pigmentation gene network was altered in Drosophila prostipennis, a species in the Drosophila melanogaster subgroup, that evolved expanded abdominal pigmentation. This expansion involved broadened expression of the melanin-promoting enzyme genes tan and yellow, and a reciprocal withdrawn pattern of the melanin-suppressing enzyme gene ebony. To examine whether these coordinated changes to the network were generated through mutations in the cis-regulatory elements (CREs) of these genes, CREs were cloned and tested of D. prostipennis tan, ebony, and yellow in transgenic reporter assays. Regulatory regions of both tan and ebony failed to recapitulate the derived D. prostipennis expression phenotype, implicating the modification of a factor or factors upstream of both genes. However, the D. prostipennis yellow cis-regulatory region recapitulated the expanded expression pattern observed in this species, implicating causative mutations in cis to yellow. The results provide an example in which a coordinated expression program evolved through independent changes at multiple loci, rather than through changes to a single 'master regulator' directing a suite of downstream target genes. This implies a complex network structure in which each gene may be subject to a unique set of inputs, and resultantly may require individualized evolutionary paths to yield the correlated gene expression patterns.
Thursday, July 3rd
Das, C., Roy, S., Namjoshi, S., Malarkey, C. S., Jones, D. N., Kutateladze, T. G., Churchill, M. E. and Tyler, J. K. (2014). Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation. Proc Natl Acad Sci U S A 111: E1072-1081. PubMed ID: 24616510
The multifunctional Creb-binding protein (CBP; see Drosophila CBP) protein plays a pivotal role in many critical cellular processes. This study demonstrate that the bromodomain of CBP binds to histone H3 (see Drosophila Histone H3) acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. Autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and it is proposed that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1; see Drosophila ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP.
Blackledge, N. P., et al. (2014). Variant PRC1 Complex-Dependent H2A Ubiquitylation Drives PRC2 Recruitment and Polycomb Domain Formation. Cell 157: 1445-1459. PubMed ID: 24856970
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. In this study, using a de novo targeting assay in mouse embryonic stem cells, it was unexpectedly discovered that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by Lysine (K)-Specific Demethylase 2B (KDM2B) to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.
Wang, Y. L., Duttke, S. H., Chen, K., Johnston, J., Kassavetis, G. A., Zeitlinger, J. and Kadonaga, J. T. (2014). TRF2, but not TBP, mediates the transcription of ribosomal protein genes. Genes Dev [Epub ahead of print]. PubMed ID: 24958592
The TCT core promoter element is present in most ribosomal protein (RP) genes in Drosophila and humans. This study shows that TBP (TATA box-binding protein)-related factor TRF2, but not TBP, is required for transcription of the TCT-dependent RP genes. In cells, TCT-dependent transcription, but not TATA-dependent transcription, increases or decreases upon overexpression or depletion of TRF2. In vitro, purified TRF2 activates TCT but not TATA promoters. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) experiments revealed the preferential localization of TRF2 at TCT versus TATA promoters. Hence, a specialized TRF2-based RNA polymerase II system functions in the synthesis of RPs and complements the RNA polymerase I and III systems.
Lhoumaud, P., Hennion, M., Gamot, A., Cuddapah, S., Queille, S., Liang, J., Micas, G., Morillon, P., Urbach, S., Bouchez, O., Severac, D., Emberly, E., Zhao, K. and Cuvier, O. (2014). Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes. EMBO J [Epub ahead of print]. PubMed ID: 24916307
Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. This study shows that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. These results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. These data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.
Wednesday, July 2nd
Mendoza, E., Colomb, J., Rybak, J., Pfluger, H. J., Zars, T., Scharff, C. and Brembs, B. (2014). Drosophila FoxP Mutants Are Deficient in Operant Self-Learning. PLoS One 9: e100648. PubMed ID: 24964149
Intact function of the Forkhead Box P2 (FOXP2) gene is necessary for normal development of speech and language. This important role has recently been extended, first to other forms of vocal learning in animals and then also to other forms of motor learning. The homology in structure and in function among the FoxP gene members raises the possibility that the ancestral FoxP gene may have evolved as a crucial component of the neural circuitry mediating motor learning. This study reports that genetic manipulations of the single Drosophila orthologue, dFoxP (forkhead domain 59A), disrupt operant self-learning, a form of motor learning sharing several conceptually analogous features with language acquisition. Structural alterations of the dFoxP locus uncovered the role of dFoxP in operant self-learning and habit formation, as well as the dispensability of dFoxP for operant world-learning, in which no motor learning occurs. These manipulations also led to subtle alterations in the brain anatomy, including a reduced volume of the optic glomeruli. RNAi-mediated interference with dFoxP expression levels copied the behavioral phenotype of the mutant flies, even in the absence of mRNA degradation. These results provide evidence that motor learning and language acquisition share a common ancestral trait still present in extant invertebrates, manifest in operant self-learning. This ‘deep’ homology probably traces back to before the split between vertebrate and invertebrate animals.
Schoofs, A., Huckesfeld, S., Schlegel, P., Miroschnikow, A., Peters, M., Zeymer, M., Spiebeta, R., Chiang, A. S. and Pankratz, M. J. (2014). Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain. PLoS Biol 12: e1001893. PubMed ID: 24960360
Central mechanisms by which specific motor programs are selected to achieve meaningful behaviors are not well understood. Using electrophysiological recordings from pharyngeal nerves upon central activation of neurotransmitter-expressing cells, this study showed that distinct neuronal ensembles can regulate different feeding motor programs. In behavioral and electrophysiological experiments, activation of 20 neurons in the brain expressing the neuropeptide hugin, a homolog of mammalian neuromedin U, simultaneously suppressed the motor program for food intake while inducing the motor program for locomotion. Decreasing hugin neuropeptide levels in the neurons by RNAi prevented this action. Reducing the level of hugin neuronal activity alone did not have any effect on feeding or locomotion motor programs. Furthermore, use of promoter-specific constructs that labeled subsets of hugin neurons demonstrated that initiation of locomotion can be separated from modulation of its motor pattern. These results provide insights into a neural mechanism of how opposing motor programs can be selected in order to coordinate feeding and locomotive behaviors.
Masek, P., Reynolds, L. A., Bollinger, W. L., Moody, C., Mehta, A., Murakami, K., Yoshizawa, M., Gibbs, A. G. and Keene, A. C. (2014). Altered regulation of sleep and feeding contribute to starvation resistance in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 24948636
Animals respond to changes in food availability by adjusting sleep and foraging strategies to optimize their fitness. Wild populations of the fruit fly, Drosophila melanogaster, display highly variable levels of starvation resistance that are dependent on geographic location, food availability, and evolutionary history. How behaviors that include sleep and feeding vary in Drosophila with increased starvation resistance is unclear. This study has generated starvation resistant flies through experimental evolution to investigate the relationship between foraging behaviors and starvation resistance. Outbred populations of D. melanogaster were selected for starvation resistance over 60 generations. This selection process resulted in flies with a three-fold increase in total lipids that survive up to 18 days without food.Starvation-selected (S) flies were tested for sleep and feeding behaviors to determine the effect that selection for starvation resistance has had on foraging behavior. Flies from three replicated starvation-selected populations displayed a dramatic reduction in feeding and prolonged sleep duration compared to fed control (F) populations, suggesting that modified sleep and feeding may contribute to starvation resistance. A prolonged larval developmental period contributes to the elevated energy stores present in starvation-selected flies. By preventing S larvae from feeding longer than F larvae, it was possible to reduce energy stores in adult S flies to levels seen in adult F flies, thus allowing control energy storage levels. However, the reduction of energy stores in S flies fails to generate normal sleep and feeding behavior seen in F flies with similar energy stores. These findings suggest that the behavioral changes observed in S flies are due to genetic regulation of behavior rather than elevated lipid levels. Testing S-F hybrid individuals for both feeding and sleep revealed a lack of correlation between food consumption and sleep duration, indicating further independence in genetic factors underlying the sleep and feeding changes observed in S flies. Taken together, these findings provide evidence that starvation selection results in prolonged sleep and reduced feeding through a mechanism that is independent of elevated energy stores. These findings suggest changes in both metabolic function and behavior contribute to the increase in starvation resistance seen in flies selected for starvation resistance.
Chihara, T., Kitabayashi, A., Morimoto, M., Takeuchi, K., Masuyama, K., Tonoki, A., Davis, R. L., Wang, J. W. and Miura, M. (2014). Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila. PLoS Genet 10: e1004437. PubMed ID: 24967585
Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity; see Death caspase-1), this study mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. It was also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.
Tuesday July 1st
Gardiol, A. and St Johnston, D. (2014). Staufen targets coracle mRNA to Drosophila neuromuscular junctions and regulates GluRIIA synaptic accumulation and bouton number. Dev Biol. PubMed ID: 24951879
The post-synaptic translation of localised mRNAs has been postulated to underlie several forms of plasticity at vertebrate synapses, but the mechanisms that target mRNAs to these postsynaptic sites are not well understood. This study shows that the evolutionary conserved dsRNA binding protein, Staufen, localises to the postsynaptic side of the Drosophila neuromuscular junction (NMJ), where it is required for the localisation of coracle mRNA and protein. Staufen plays a well-characterised role in the localisation of oskar mRNA to the oocyte posterior, where Staufen dsRNA-binding domain 5 is specifically required for its translation. Removal of Staufen dsRNA-binding domain 5, disrupts the postsynaptic accumulation of Coracle protein without affecting the localisation of cora mRNA, suggesting that Staufen similarly regulates Coracle translation. Tropomyosin II, which functions with Staufen in oskar mRNA localisation, is also required for coracle mRNA localisation, suggesting that similar mechanisms target mRNAs to the NMJ and the oocyte posterior. Coracle, the orthologue of vertebrate band 4.1, functions in the anchoring of the glutamate receptor IIA subunit (GluRIIA) at the synapse. Consistent with this, staufen mutant larvae show reduced accumulation of GluRIIA at synapses. The NMJs of staufen mutant larvae have also a reduced number of synaptic boutons. Altogether, this suggests that this novel Staufen-dependent mRNA localisation and local translation pathway may play a role in the developmentally regulated growth of the NMJ.
Koch, N., Kobler, O., Thomas, U., Qualmann, B. and Kessels, M. M. (2014). Terminal axonal arborization and synaptic bouton formation critically rely on abp1 and the arp2/3 complex. PLoS One 9: e97692. PubMed ID: 24841972
Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. This study shows that the F-actin-binding protein Abp1 is prominently expressed in the Drosophila nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. Abp1 knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied abp1 knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. These data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.
Liu, H., Zhou, B., Yan, W., Lei, Z., Zhao, X., Zhang, K. and Guo, A. (2014). Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila. Eur J Neurosci [Epub ahead of print]. PubMed ID: 24964821
Astrocyte-like glial cells are abundant in the central nervous system of adult Drosophila and exhibit morphology similar to astrocytes of mammals. Previous evidence has shown that astrocyte-like glial cells are strongly associated with synapses in the antennal lobe (AL), the first relay of the olfactory system, where olfactory receptor neurons (ORNs) transmit information into projection neurons (PNs). However, the function of astrocyte-like glia in the AL remains obscure. This study used in vivo calcium imaging to find that astrocyte-like glial cells exhibit spontaneous microdomain calcium elevations. Using simultaneous manipulation of glial activity and monitoring of neuronal function, it was found that the astrocyte-like glial activation, but not ensheathing glial activation, could inhibit odor-evoked responses of PNs. Ensheathing glial cells are another subtype of glia, and are of functional importance in the AL. Electrophysiological experiments indicated that astrocyte-like glial activation decreases the amplitude and slope of excitatory postsynaptic potentials evoked through electrical stimulation of the antennal nerve. These results suggest that astrocyte-like glial cells may regulate olfactory processing through negative regulation of ORN-PN synaptic strength. Beyond the antennal lobe astrocyte-like glial spontaneous calcium activities were observed in the ventromedial protocerebrum, indicating that astrocyte-like glial spontaneous calcium elevations might be general in the adult fly brain. Overall, this study demonstrates a new function for astrocyte-like glial cells in the physiological modulation of olfactory information transmission, possibly through regulating ORN-PN synapse strength.
Liu, H., Bai, H., Xue, R., Takahashi, H., Edwardson, J. M. and Chapman, E. R. (2014). Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission. Nat Neurosci 17: 670-677. PubMed ID: 24657966
The Ca(2+) sensor for rapid synaptic vesicle exocytosis, synaptotagmin 1 (syt) (see Drosophila Synaptotagmin 1), is largely composed of two Ca(2+)-sensing C2 domains, C2A and C2B. This study investigated the apparent synergy between the tandem C2 domains by altering the length and rigidity of the linker that connects them. The behavior of the linker mutants revealed a correlation between the ability of the C2 domains to penetrate membranes in response to Ca(2+) and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Using atomic force microscopy, this study found that the synergy between these C2 domains involved intra-molecular interactions between them. Thus, syt function is markedly affected by changes in the physical nature of the linker that connects its tandem C2 domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp before the Ca(2+) trigger.
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