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Monday, March 31, 2014

Niewiadomski, P., Kong, J. H., Ahrends, R., Ma, Y., Humke, E. W., Khan, S., Teruel, M. N., Novitch, B. G. and Rohatgi, R. (2014). Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. Cell Rep 6: 168-181. PubMed ID: 24373970
Summary: Gli proteins (Drosophila homolog: Cubitus interruptus) are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. This paper describes a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA; see Drosophila PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity.

Takats, S., Pircs, K., Nagy, P., Varga, A., Karpati, M., Hegedus, K., Kramer, H., Kovacs, A. L., Sass, M. and Juhasz, G. (2014). Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol Cell [Epub ahead of print]. PubMed ID: 24554766
Summary: HOPS is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. This study shows that all six HOPS subunits (Vps11/CG32350, Vps18/Deep Orange, Vps16A, Vps33A/Carnatine, Vps39/CG7146 and Vps41/Light) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced and developmental conditions. HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting that their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes, and for proper trafficking to lysosomes and eye pigment granules. It was also shown that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG, a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. These results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.

Zhang, J., Du, J., Lei, C., Liu, M. and Zhu, A. J. (2014). Ubpy controls the stability of the ESCRT-0 subunit Hrs in development. Development 141(7): 1473-9. PubMed ID: 24574010
Summary: Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. This study shows that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. In the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. It was further demonstrated that this phenotype results from endocytic pathway defects. Evidence is provided that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, it was found that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, these data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development.

Lin, C., Koval, A., Tishchenko, S., Gabdulkhakov, A., Tin, U., Solis, G. P. and Katanaev, V. L. (2014). Double Suppression of the Galpha Protein Activity by RGS Proteins. Mol Cell 53: 663-671. PubMed ID: 24560274
Summary: Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis on G protein alpha subunits, restricting their activity downstream from G protein-coupled receptors. This study has identified Drosophila Double hit (Dhit) as a dual RGS regulator of Galphao. In addition to the conventional GTPase-activating action, Dhit possesses the guanine nucleotide dissociation inhibitor (GDI) activity, slowing the rate of GTP uptake by Galphao; both activities are mediated by the same RGS domain. These findings are recapitulated using homologous mammalian Galphao/i proteins and RGS19. Crystal structure and mutagenesis studies provide clues into the molecular mechanism for this unprecedented GDI activity. Physiologically, this activity was cofirmed in Drosophila asymmetric cell divisions and HEK293T cells. The oncogenic Galphao mutant found in breast cancer escapes this GDI regulation. These studies identify Dhit and its homologs as double-action regulators, inhibiting Galphao/i proteins both through suppression of their activation and acceleration of their inactivation through the single RGS domain.

Sunday, March 30th

Messina, G., et al. (2014). Yeti, a Drosophila melanogaster essential gene, encodes a protein required for chromatin organization. J Cell Sci [Epub ahead of print]. PubMed ID: 24652835
Summary: The evolutionarily conserved family of Bucentaur (BCNT) proteins exhibits a widespread distribution in animal and plants, yet its biological role remains largely unknown. Using Drosophila melanogaster as a model organism, This study investigated the in vivo role of the Drosophila BCNT member called Yeti. Loss of Yeti causes lethality before pupation and defects in higher order chromatin organization, evidenced by severe impairment in the association of histone H2A.V, nucleosomal histones and epigenetic marks with polytene chromosomes. Yeti binds to polytene chromosomes through its conserved BCNT domain and interacts with the histone variant H2A.V, HP1a and Domino-A (DOM-A), the ATPase subunit of the DOM/Tip60 chromatin remodeling complex. Furthermore, Yeti was identified as a novel downstream target of the Drosophila DOM-A. Based on these results, it is proposed that Yeti interacts with H2A.V-exchanging machinery, as a chaperone or as a new subunit of the DOM/Tip60 remodeling complex, and contributes to regulate the accumulation of H2A.V at chromatin sites. Overall, these findings suggest an unanticipated role of Yeti protein in chromatin organization and provide for the first time mechanistic clues on how BCNT proteins may control development in multicellular organisms.

Satyaki, P. R., Cuykendall, T. N., Wei, K. H., Brideau, N. J., Kwak, H., Aruna, S., Ferree, P. M., Ji, S. and Barbash, D. A. (2014). The hmr and lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats. PLoS Genet 10: e1004240. PubMed ID: 24651406
Summary: Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. This study reports that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses it was discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans several satellite DNAs and TEs were identified that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. It as further found that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. These results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.

Kusch, T., Mei, A. and Nguyen, C. (2014). Histone H3 lysine 4 trimethylation regulates cotranscriptional H2A variant exchange by Tip60 complexes to maximize gene expression. Proc Natl Acad Sci U S A. PubMed ID: 24639513
Summary: Histone H3 lysine 4 trimethylation (H3K4me3) and the acetylated H2A variant, H2A.Z/v (H2Avac), are enriched at promoters of highly transcribed loci including the stress response genes. Using the inducible Drosophila hsp70 loci as a model, the roles of the dSet1 and dTip60 complexes in the generation of these two chromatin modifications was studied. Heat Shock Factor was found to recruit the dTip60 complex to the hsp70 loci in cells treated with salicylate, which triggers chromatin remodeling at these loci without transcription activation. Under these conditions, H2Avac or H3K4me3 are not enriched at the hsp70 promoter. By contrast, heat shock-induced hsp70 transcription induces dSet1-dependent H3K4me3 and H2Avac deposition by the dTip60 complex. The loss of dSet1 or dTip60 abolishes H2Avac incorporation, impairs Pol II release from the hsp70 promoter, and causes a stalling of mRNA production during phases of transcription maximization. Biochemical assays confirm that nucleosomal H3K4me3 stimulates the histone acetyltransferase and H2Av exchange activities of dTip60 complexes. H2Avac contributes to nucleosome destabilization at promoters, and H3K4me3 restricts its incorporation to phases of acute transcription. The process uncouples cotranscriptional chromatin remodeling by dTip60 complexes from their role in the activation of PARP, which is responsible for the removal of transcription-incompatible or damaged chromatin during the initial stress response. The control of the multifunctional dTip60 complex by H3K4me3 ensures optimal stress response and cell survival by mediating the rapid maximization of hsp70 expression. Furthermore, this mechanism prevents the accumulation of epigenetic noise caused by random complex-nucleosome collisions.

Zielke, T. and Saumweber, H. (2014). Dissection of open chromatin domain formation by site specific recombination in Drosophila. J Cell Sci [Epub ahead of print]. PubMed ID: 24639466
Summary: Drosophila polytene interphase chromosomes provide an ideal test system to study the rules that define the structure of chromatin domains. A transgenic condensed chromatin domain cassette was established for the insertion of large pieces of DNA by site specific recombination. Insertion of this cassette into open chromatin generated a condensed domain, visible as an extra band on polytene chromosomes. Site specific recombination of DNA sequence variants into this ectopic band allowed comparison of their capacity for open chromatin formation by cytogenetic methods. The 61C7-8 interband DNA was demonstrated to maintain its open chromatin conformation and epigenetic state at an ectopic position. By deletion analysis the sequences essential for open chromatin formation was mapped to a 490 bp fragment in the proximal part of the 17 kb interband sequence. This fragment overlaps binding sites of the chromatin protein Chriz, the histone kinase Jil-1 and the boundary element protein CP190. It also overlaps a promoter region that locates in between the Rev1 and Med30 transcription units.

Saturday, March 29th

Andrade-Zapata, I. and Baonza, A. (2014). The bHLH Factors Extramacrochaetae and Daughterless Control Cell Cycle in Drosophila Imaginal Discs through the Transcriptional Regulation of the cdc25 Phosphatase string. PLoS Genet 10: e1004233. PubMed ID: 24651265
Summary: One of the major issues in developmental biology is about having a better understanding of the mechanisms that regulate organ growth. Identifying these mechanisms is essential to understand the development processes that occur both in physiological and pathological conditions, such as cancer. The E protein family of basic helix-loop helix (bHLH) transcription factors, and their inhibitors the Id proteins, regulate cell proliferation in metazoans. This notion is further supported because the activity of these factors is frequently deregulated in cancerous cells. The E protein orthologue Daughterless (Da) and the Id orthologue Extramacrochaetae (Emc) are the only members of these classes of bHLH proteins in Drosophila. Although these factors are involved in controlling proliferation, the mechanism underlying this regulatory activity is poorly understood. Through a genetic analysis, this study shows that during the development of epithelial cells in the imaginal discs, the G2/M transition, and hence cell proliferation, is controlled by Emc via Da. In eukaryotic cells, the main activator of this transition is the Cdc25 phosphatase, string. Genetic analyses reveal that the ectopic expression of string in cells with reduced levels of Emc or high levels of Da is sufficient to rescue the proliferative defects seen in these mutant cells. Moreover, evidence is presented demonstrating a role of Da as a transcriptional repressor of string. Taken together, these findings define a mechanism through which Emc controls cell proliferation by regulating the activity of Da, which transcriptionally represses string.

Choe, S. K., Ladam, F. and Sagerstrom, C. G. (2014). TALE factors poise promoters for activation by Hox proteins. Dev Cell 28: 203-211. PubMed ID: 24480644
Summary: Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis (see Drosophila Extradenticle and Homothorax) families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a (Drosophila homolog labial) transcription, this study found maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb (see Drosophila Cdk9, the co-factor of Drosophila P-TEFb). However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. It is concludes that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription.

Xu, Z., Chen, H., Ling, J., Yu, D., Struffi, P. and Small, S. (2014). Impacts of the ubiquitous factor Zelda on Bicoid-dependent DNA binding and transcription in Drosophila. Genes Dev 28: 608-621. PubMed ID: 24637116
Summary: In vivo cross-linking studies suggest that the Drosophila transcription factor Bicoid (Bcd) binds to several thousand sites during early embryogenesis, but it is not clear how many of these binding events are functionally important. In contrast, reporter gene studies have identified >60 Bcd-dependent enhancers, all of which contain clusters of the consensus binding sequence TAATCC. These studies also identified clusters of TAATCC motifs (inactive fragments) that failed to drive Bcd-dependent activation. In general, active fragments showed higher levels of Bcd binding in vivo and were enriched in predicted binding sites for the ubiquitous maternal protein Zelda (Zld). This study tested the role of Zld in Bcd-mediated binding and transcription. Removal of Zld function and mutations in Zld sites caused significant reductions in Bcd binding to known enhancers and variable effects on the activation and spatial positioning of Bcd-dependent expression patterns. Also, insertion of Zld sites converted one of six inactive fragments into a Bcd-responsive enhancer. Genome-wide binding experiments in zld mutants showed variable effects on Bcd-binding peaks, ranging from strong reductions to significantly enhanced levels of binding. Increases in Bcd binding caused the precocious Bcd-dependent activation of genes that are normally not expressed in early embryos, suggesting that Zld controls the genome-wide binding profile of Bcd at the qualitative level and is critical for selecting target genes for activation in the early embryo. These results underscore the importance of combinatorial binding in enhancer function and provide data that will help predict regulatory activities based on DNA sequence.

Zehavi, Y., Kuznetsov, O., Ovadia-Shochat, A. and Juven-Gershon, T. (2014. Core Promoter Functions in the Regulation of Gene Expression of Drosophila Dorsal Target Genes. J Biol Chem [Epub ahead of print]. PubMed ID: 24634215
Summary: Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the Inr (initiator), TATA box and DPE (downstream core promoter element), which confer specific properties to the core promoter. This study explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NF kappaB-homolog transcription factor, along the dorsal-ventral axis. Over two thirds of Dorsal target genes were shown to contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. Multiple Dorsal target genes were shown to be evolutionarily conserved and functionally dependent on the DPE. Furthermore, the activation of key Dorsal target genes by Dorsal was studied, as well as by another Rel family transcription factor, Relish, along with the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, it was demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, these results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes.

Friday, March 28th

Seluzicki, A., Flourakis, M., Kula-Eversole, E., Zhang, L., Kilman, V. and Allada, R. (2014). Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior. PLoS Biol 12: e1001810. PubMed ID: 24643294
Summary: Molecular circadian clocks are interconnected via neural networks. In Drosophila, Pigment-Dispersing Factor (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF+ and PDF- circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. This study has demonstrated that genetic inhibition of protein kinase A (PKA) in PDF- clock neurons can phenocopy PDF mutants, while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, per was rescued in PDF neurons of arrhythmic per01 mutants. PDF neuron rescue induced high amplitude rhythms in the clock component Timeless (Tim) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced Tim levels in non-PDF neurons of per01 flies. To address how PDF impacts pacemaker neuron output, PDF was focally applied to DN1p neurons and was found to acutely depolarize and increase firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. Evidence is provided for a signaling mechanism (PKA) and a molecular target (Tim) by which PDF resets and synchronizes clocks; PDF exhibits an acute direct excitatory effect on target neurons to control neuronal output. The identification of Tim as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, these results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output.

Liu, Y. C., Pearce, M. W., Honda, T., Johnson, T. K., Charlu, S., Sharma, K. R., Imad, M., Burke, R. E., Zinsmaier, K. E., Ray, A., Dahanukar, A., de Bruyne, M. and Warr, C. G. (2014). The Drosophila melanogaster Phospholipid Flippase dATP8B Is Required for Odorant Receptor Function. PLoS Genet 10: e1004209. PubMed ID: 24651716
Summary: The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR; see for example Ionotropic receptor 84a) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. This study reports the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B (CG14741), the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, dATP8B was found to be expressed in ORNs and localises to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, these findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins.

Rezaval, C., Nojima, T., Neville, M. C., Lin, A. C. and Goodwin, S. F. (2014). Sexually Dimorphic Octopaminergic Neurons Modulate Female Postmating Behaviors in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 24631243
Summary: Mating elicits profound behavioral and physiological changes in many species that are crucial for reproductive success. After copulation, Drosophila melanogaster females reduce their sexual receptivity and increase egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via SP sensory neurons in the uterus defined by coexpression of the proprioceptive neuronal marker pickpocket (ppk) and the sex-determination genes doublesex (dsx) and fruitless (fru). Although neurons expressing dsx downstream of SP signaling have been shown to regulate postmating behaviors, how the female nervous system coordinates the change from pre- to postcopulatory states is unknown. This study shows a role of the neuromodulator octopamine (OA) in the female postmating response. Lack of OA disrupts postmating responses in mated females, while increase of OA induces postmating responses in virgin females. Using a novel dsxFLP allele, dsx neuronal elements were uncovered associated with OA signaling involved in modulation of postmating responses. A small subset of sexually dimorphic OA/dsx+ neurons (approximately nine cells in females) were identified in the abdominal ganglion. These results are consistent with a model whereby OA neuronal signaling increases after copulation, which in turn modulates changes in female behavior and physiology in response to reproductive state.

Heifetz, Y., Lindner, M., Garini, Y. and Wolfner, M. F. (2014). Mating Regulates Neuromodulator Ensembles at Nerve Termini Innervating the Drosophila Reproductive Tract. Curr Biol [Epub ahead of print]. PubMed ID: 24631240
Summary: Upon mating, regions of the female reproductive tract mature and alter their function, for example to facilitate storage of sperm or control the release of eggs. The female's nervous system and neuromodulators play important roles in her responses to mating. However, it is difficult to reconcile the reproductive tract's many changing but coordinated events with the small set of neuromodulators present. It was hypothesized that each part of the reproductive tract contains a characteristic combination of neuromodulators that confer unique identities on each region and that postmating changes in these combinations coordinate subsequent actions. The presence, locations, and levels of neuromodulators and related molecules ('signaling molecules') was examined in the reproductive tract of Drosophila melanogaster females before and after mating: the biogenic amine octopamine, which regulates ovulation rate in Drosophila and locusts; serotonin, which regulates muscle contraction in locust oviducts; and the FMRF amide dromyosuppressin, which regulates contraction of Drosophila heart muscle and may regulate muscle contractions in the reproductive tract, if it is expressed there. It was found that separate aspects of mating (sperm, seminal proteins, and physical effects) independently modulate the release of signaling molecules. Each reproductive tract subregion displays a characteristic combination of signaling molecule release, resulting in a unique functional identity. These patterns, and thus functions, change reproducibly after mating. Thus, one event (mating) promotes new combinations of signaling molecules that endow different parts of the reproductive tract with unique temporal and spatial identities that facilitate many aspects of fertilization.

Thursday, March 27th

Zheng, X., Valakh, V., Diantonio, A. and Ben-Shahar, Y. (2014). Natural antisense transcripts regulate the neuronal stress response and excitability. Elife 3: e01849. PubMed ID: 24642409
Summary: Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. This study shows that pickpocket 29 (ppk29), a gene that encodes a Drosophila degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. The mRNA 3'UTR of ppk29 affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of seizure (sei), the Drosophila homolog of the human Ether-a-go-go Related Gene (hERG) potassium channel. The regulatory impact of ppk29 mRNA on sei is independent of the sodium channel it encodes. Thus, these studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity.

Geiger, J. C., Lipka, J., Segura, I., Hoyer, S., Schlager, M. A., Wulf, P. S., Weinges, S., Demmers, J., Hoogenraad, C. C. and Acker-Palmer, A. (2014). The GRIP1/14-3-3 Pathway Coordinates Cargo Trafficking and Dendrite Development. Dev Cell 28: 381-393. PubMed ID: 24576423
Summary: Regulation of cargo transport via adaptor molecules is essential for neuronal development. However, the role of PDZ scaffolding proteins as adaptors in neuronal cargo trafficking is still poorly understood. This study shows by genetic deletion in mice that the multi-PDZ domain scaffolding protein glutamate receptor interacting protein 1 (GRIP1) is required for dendrite development. An interaction was identified between GRIP1 and 14-3-3 proteins (see Drosophila 14-3-3zeta) that is essential for the function of GRIP1 as an adaptor protein in dendritic cargo transport. Mechanistically, 14-3-3 binds to the kinesin-1 binding region in GRIP1 in a phospho-dependent manner and detaches GRIP1 from the kinesin-1 (see Drosophila Kinesin heavy chain) motor protein complex thereby regulating cargo transport. A single point mutation in the Thr956 of GRIP1 in transgenic mice impairs dendritic development. Together, these results show a regulatory role for GRIP1 during microtubule-based transport and suggest a crucial function for 14-3-3 proteins in controlling kinesin-1 motor attachment during neuronal development.

Cheng, T. L., Wang, Z., Liao, Q., Zhu, Y., Zhou, W. H., Xu, W. and Qiu, Z. (2014). MeCP2 Suppresses Nuclear MicroRNA Processing and Dendritic Growth by Regulating the DGCR8/Drosha Complex. Dev Cell 28: 547-560. PubMed ID: 24636259
Summary: Loss- and gain-of-function mutations of the X-linked gene MECP2 (methyl-CpG binding protein 2) lead to severe neurodevelopmental disorders in humans, such as Rett syndrome (RTT) and autism. MeCP2 is previously known as a transcriptional repressor by binding to methylated DNA and recruiting histone deacetylase complex (HDAC). This study reports that MeCP2 regulates gene expression posttranscriptionally by suppressing nuclear microRNA processing. MeCP2 was found to bind directly to DiGeorge syndrome critical region 8 (DGCR8; Drosophila homolog, Partner of Drosha), a critical component of the nuclear microRNA-processing machinery, and interferes with the assembly of Drosha and DGCR8 complex. Protein targets of MeCP2-suppressed microRNAs include CREB, LIMK1, and Pumilio2, which play critical roles in neural development. Gain of function of MeCP2 strongly inhibits dendritic and spine growth, which depends on the interaction of MeCP2 and DGCR8. Thus, control of microRNA processing via direct interaction with DGCR8 represents a mechanism for MeCP2 regulation of gene expression and neural development.

Wittmann, W., Iulianella, A. and Gunhaga, L. (2014). Cux2 acts as a critical regulator for neurogenesis in the olfactory epithelium of vertebrates. Dev Biol 388: 35-47. PubMed ID: 24512687
Summary: Signaling pathways and transcription factors are crucial regulators of vertebrate neurogenesis, exerting their function in a spatial and temporal manner. Despite recent advances in understanding of the molecular regulation of embryonic neurogenesis, little is known regarding how different signaling pathways interact to tightly regulate this process during the development of neuroepithelia. This study has investigated the events lying upstream and downstream of a key neurogenic factor, the Cut-like homeodomain transcription factor-2 (Cux2; see Drosophila Cut), during embryonic neurogenesis in chick and mouse. By using the olfactory epithelium as a model for neurogenesis this study analyzed mouse embryos deficient in Cux2, as well as chick embryos exposed to Cux2 silencing (si) RNA or a Cux2 over-expression construct. Evidence is provided that enhanced BMP activity increases Cux2 expression and suppresses olfactory neurogenesis in the chick olfactory epithelium. In addition, the results show that up-regulation of Cux2, either BMP-induced or ectopically over-expressed, reduces Delta1 expression and suppresses proliferation. Interestingly, the loss of Cux2 activity, using mutant mice or siRNA in chick, also diminishes neurogenesis, Notch activity and cell proliferation in the olfactory epithelium. The results suggest that controlled low levels of Cux2 activity are necessary for proper Notch signaling, maintenance of the proliferative pool and ongoing neurogenesis in the olfactory epithelium. Thus, this study demonstrates a novel conserved mechanism in vertebrates in which levels of Cux2 activity play an important role for ongoing neurogenesis in the olfactory epithelium.

Wednesday, March 26th

Wu, M., Robinson, J. E. and Joiner, W. J. (2014). SLEEPLESS Is a Bifunctional Regulator of Excitability and Cholinergic Synaptic Transmission. Curr Biol 24(6): 621-9. PubMed ID: 24613312
Summary: Although sleep is conserved throughout evolution, the molecular basis of its control is still largely a mystery. It has been previously showen that the quiver/sleepless (qvr/sss) gene encodes a membrane-tethered protein that is required for normal sleep in Drosophila. SLEEPLESS (SSS) protein functions, at least in part, by upregulating the levels and open probability of Shaker (Sh) potassium channels to suppress neuronal excitability and enable sleep. Consistent with this proposed mechanism, loss-of-function mutations in Sh phenocopy qvr/sss-null mutants. However, sleep is more genetically modifiable in Sh than in qvr/sss mutants, suggesting that SSS may regulate additional molecules to influence sleep. This study shows that SSS also antagonizes nicotinic acetylcholine receptors (nAChRs) to reduce synaptic transmission and promote sleep. Mimicking this antagonism with the nAChR inhibitor mecamylamine or by RNAi knockdown of specific nAChR subunits is sufficient to restore sleep to qvr/sss mutants. Regulation of nAChR activity by SSS occurs posttranscriptionally, since the levels of nAChR mRNAs are unchanged in qvr/sss mutants. Regulation of nAChR activity by SSS may in fact be direct, since SSS forms a stable complex with and antagonizes nAChR function in transfected cells. Intriguingly, lynx1, a mammalian homolog of SSS, can partially restore normal sleep to qvr/sss mutants, and Lynx1 can form stable complexes with Shaker-type channels and nAChRs. These data point to an evolutionarily conserved, bifunctional role for SSS and its homologs in controlling excitability and synaptic transmission in fundamental processes of the nervous system such as sleep.

Joiner, W. J., Friedman, E. B., Hung, H. T., Koh, K., Sowcik, M., Sehgal, A. and Kelz, M. B. (2013). Genetic and anatomical basis of the barrier separating wakefulness and anesthetic-induced unresponsiveness. PLoS Genet 9: e1003605. PubMed ID: 24039590
Summary: A robust, bistable switch regulates the fluctuations between wakefulness and natural sleep as well as those between wakefulness and anesthetic-induced unresponsiveness.Experimental evidence has been provided for the existence of a behavioral barrier to transitions between these states of arousal, which are called neural inertia. This study shows that neural inertia is controlled by processes that contribute to sleep homeostasis and requires four genes involved in electrical excitability: Sh, sss, na and unc79. Although loss of function mutations in these genes can increase or decrease sensitivity to anesthesia induction, surprisingly, they all collapse neural inertia. These effects are genetically selective: neural inertia is not perturbed by loss-of-function mutations in all genes required for the sleep/wake cycle. These effects are also anatomically selective: sss acts in different neurons to influence arousal-promoting and arousal-suppressing processes underlying neural inertia. Supporting the idea that anesthesia and sleep share some, but not all, genetic and anatomical arousal-regulating pathways, it was demonstrated that increasing homeostatic sleep drive widens the neural inertial barrier. It is proposed that processes selectively contributing to sleep homeostasis and neural inertia may be impaired in pathophysiological conditions such as coma and persistent vegetative states.

Chen, W. F., Maguire, S., Sowcik, M., Luo, W., Koh, K. and Sehgal, A. (2014). A neuron-glia interaction involving GABA transaminase contributes to sleep loss in sleepless mutants. Mol Psychiatry [Epub ahead of print]. PubMed ID: 24637426
Summary: Sleep is an essential process and yet mechanisms underlying it are not well understood. Loss of the Drosophila quiver/sleepless (qvr/sss) gene increases neuronal excitability and diminishes daily sleep, providing an excellent model for exploring the underpinnings of sleep regulation. This study used a proteomic approach to identify proteins altered in sss brains. Loss of sleepless post-transcriptionally elevates the CG7433 protein, a mitochondrial gamma-aminobutyric acid transaminase (GABAT), and reduces GABA in fly brains. Loss of GABAT increases daily sleep and improves sleep consolidation, indicating that GABAT promotes wakefulness. Importantly, disruption of the GABAT gene completely suppresses the sleep phenotype of sss mutants, demonstrating that GABAT is required for loss of sleep in sss mutants. While SSS acts in distinct populations of neurons, GABAT acts in glia to reduce sleep in sss flies. These results identify a novel mechanism of interaction between neurons and glia that is important for the regulation of sleep.

Liu, S., Lamaze, A., Liu, Q., Tabuchi, M., Yang, Y., Fowler, M., Bharadwaj, R., Zhang, J., Bedont, J., Blackshaw, S., Lloyd, T. E., Montell, C., Sehgal, A., Koh, K. and Wu, M. N. (2014). WIDE AWAKE Mediates the Circadian Timing of Sleep Onset. Neuron 82(1): 151-66. PubMed ID: 24631345
Summary: How the circadian clock regulates the timing of sleep is poorly understood. This study identifies a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of WAKE in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. WAKE levels cycle, peaking near dusk, and the expression of WAKE in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring WAKE expression in LNvs. WAKE interacts with the GABAA receptor Resistant to Dieldrin (RDL), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. It is proposed that WAKE acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep.

Tuesday, March 25th

Vernes, S. C. (2014). Genome wide identification of Fruitless targets suggests a role in upregulating genes important for neural circuit formation. Sci Rep 4: 4412. PubMed ID: 24642956
Summary: The fruitless gene (fru) encodes a set of transcription factors (Fru) that display sexually dimorphic gene expression in the brain of Drosophila. Behavioural studies have demonstrated that fru is essential for courtship behaviour in the male fly and is thought to act by directing the development of sex-specific neural circuitry that encodes this innate behavioural response. This study reports the identification of direct regulatory targets of the sexually dimorphic isoforms of the Fru protein using an in vitro model system. Genome wide binding sites were identified for each of the isoforms using Chromatin Immunoprecipitation coupled to deep sequencing (ChIP-Seq). Putative target genes were found to be involved in processes such as neurotransmission, ion-channel signalling and neuron development. All isoforms showed a significant bias towards genes located on the X-chromosome, which may reflect a specific role for Fru in regulating x-linked genes. Taken together with expression analysis carried out in Fru positive neurons specifically isolated from the male fly brain, it appears that the Fru protein acts as a transcriptional activator. Understanding the regulatory cascades induced by Fru will help to shed light on the molecular mechanisms that are important for specification of neural circuitry underlying complex behaviour.

Shimono, K., Fujishima, K., Nomura, T., Ohashi, M., Usui, T., Kengaku, M., Toyoda, A. and Uemura, T. (2014). An evolutionarily conserved protein CHORD regulates scaling of dendritic arbors with body size. Sci Rep 4: 4415. PubMed ID: 24643112
Summary: Most organs scale proportionally with body size through regulation of individual cell size and/or cell number. Here we addressed how postmitotic and morphologically complex cells such as neurons scale with the body size by using the dendritic arbor of one Drosophila sensory neuron as an assay system. In small adults eclosed under a limited-nutrition condition, the wild-type neuron preserved the branching complexity of the arbor, but scaled down the entire arbor, making a 'miniature'. In contrast, mutant neurons for the Insulin/IGF signaling (IIS) or TORC1 pathway exhibited 'undergrowth', which was characterized by decreases in both the branching complexity and the arbor size, despite a normal diet. These contrasting phenotypes hinted that a novel regulatory mechanism contributes to the dendritic scaling in wild-type neurons. Indeed, this study isolated a mutation in the gene CHORD/morgana that uncoupled the neuron size and the body size: CHORD mutant neurons generated miniature dendritic arbors regardless of the body size. CHORD encodes an evolutionarily conserved co-chaperone of HSP90. The results support the notion that dendritic growth and branching are controlled by partly separate mechanisms. The IIS/TORC1 pathways control both growth and branching to avert underdevelopment, whereas CHORD together with TORC2 realizes proportional scaling of the entire arbor.

Koe, C. T., et al. (2014). The Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages. Elife 3: e01906. PubMed ID: 24618901
Summary: The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology. Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors (INPs) is unrestrained. This study demonstrates that Drosophila SWI/SNF chromatin remodeling Brahma (Brm) complex functions cooperatively with another chromatin remodeling factor, Histone deacetylase 3 (HDAC3) to suppress the formation of ectopic type II neuroblasts. Multiple components of the Brm complex and HDAC3 physically associate with Earmuff (Erm), a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts. Consistently, the predicted Erm-binding motif is present in most of known binding loci of Brm. Furthermore, brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth. Thus, the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts.

Hovhanyan, A., Herter, E. K., Pfannstiel, J., Gallant, P. and Raabe, T. (2014). Drosophila Mbm is a Nucleolar Myc and CK2 Target Required for Ribosome Biogenesis and Cell Growth of Central Brain Neuroblasts. Mol Cell Biol [Epub ahead of print]. PubMed ID: 24615015
Summary: Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy sensing organs into growth promoting processes such as the sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila. Yet, the molecular function of Mbm has been unknown so far. This study shows that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which among other functions relays information from nutrient dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2, which has an impact on localization of the protein. It is concluded that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which together with CK2-mediated signals enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth.

Monday, March 24th

Lin, A. Y. and Pearson, B. J. (2014). Planarian yorkie/YAP functions to integrate adult stem cell proliferation, organ homeostasis and maintenance of axial patterning. Development 141: 1197-1208. PubMed ID: 24523458
Summary: During adult homeostasis and regeneration, the freshwater planarian must accomplish a constant balance between cell proliferation and cell death, while also maintaining proper tissue and organ size and patterning. How these ordered processes are precisely modulated remains relatively unknown. This study shows that planarians use the downstream effector of the Hippo signaling cascade, yorkie (yki; YAP in vertebrates, Yorkie in Drosophila) to control a diverse set of pleiotropic processes in organ homeostasis, stem cell regulation, regeneration and axial patterning. yki functions to maintain the homeostasis of the planarian excretory (protonephridial) system and to limit stem cell proliferation, but does not affect the differentiation process or cell death. Finally, Yki acts synergistically with WNT/beta-catenin signaling to repress head determination by limiting the expression domains of posterior WNT genes and that of the WNT-inhibitor notum (see Drosophila Notum). Together, these data show that yki is a key gene in planarians that integrates stem cell proliferation control, organ homeostasis, and the spatial patterning of tissues.

Basta, J. M., Robbins, L., Kiefer, S. M., Dorsett, D. and Rauchman, M. (2014). Sall1 balances self-renewal and differentiation of renal progenitor cells. Development 141: 1047-1058. PubMed ID: 24550112
Summary: The formation of the proper number of functional nephrons requires a delicate balance between renal progenitor cell self-renewal and differentiation. The molecular factors that regulate the dramatic expansion of the progenitor cell pool and differentiation of these cells into nephron precursor structures (renal vesicles) are not well understood. This study shows that Sall1 (see Drosophila Spalt), a nuclear transcription factor, is required to maintain the stemness of nephron progenitor cells. Transcriptional profiling of Sall1 mutant cells revealed a striking pattern, marked by the reduction of progenitor genes and amplified expression of renal vesicle differentiation genes. These global changes in gene expression were accompanied by ectopic differentiation at E12.5 and depletion of Six2+Cited1+ cap mesenchyme progenitor cells. These findings highlight a novel role for Sall1 in maintaining the stemness of the progenitor cell pool by restraining their differentiation into renal vesicles.

Tian, A. and Jiang, J. (2014). Intestinal epithelium-derived BMP controls stem cell self-renewal in Drosophila adult midgut. Elife 3: e01857. PubMed ID: 24618900
Summary: Stem cells are maintained in a specialized microenvironment called niche but the nature of stem cell niche remains poorly defined in many systems. This study demonstrates that intestinal epithelium-derived BMP serves as a niche signal for intestinal stem cell (ISC) self-renewal in Drosophila adult midgut. BMP signaling was found to be asymmetric between ISC and its differentiated daughter cell. Two BMP ligands, Dpp and Gbb, are produced by enterocytes and act in conjunction to promote ISC self-renewal by antagonizing Notch signaling. Furthermore, the basement membrane-associated type IV collagens regulate ISC self-renewal by confining higher BMP signaling to ISCs. The employment of gut epithelia as a niche for stem cell self-renewal may provide a mechanism for direct communication between the niche and the environment, allowing niche signal production and stem cell number to be fine-tuned in response to various physiological and pathological stimuli.

Kayukawa, T., Murata, M., Kobayashi, I., Muramatsu, D., Okada, C., Uchino, K., Sezutsu, H., Kiuchi, M., Tamura, T., Hiruma, K., Ishikawa, Y. and Shinoda, T. (2014). Hormonal regulation and developmental role of Kruppel homolog 1, a repressor of metamorphosis, in the silkworm Bombyx mori. Dev Biol 388: 48-56. PubMed ID: 24508345
Summary: Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Kruppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. This study attempts to elucidate the hormonal regulation and developmental role of Kr-h1. The expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was found to be induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, transgenic silkworms were generated overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval-larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis.

Sunday, March 23rd

Jones, T. A., Nikolova, L. S., Schjelderup, A. and Metzstein, M. M. (2014). Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells. Jones, T. A., Nikolova, L. S., Schjelderup, A. and Metzstein, M. M. (2014). Dev Biol [Epub ahead of print]. PubMed ID: 24607370
Summary: Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increases surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. This study used Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs on the single cell level. The exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, was shown to be required for terminal cell branch outgrowth. Exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. Vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex (see 14-3-3zeta/Par5) is required for branching, and the PAR complex was found to be required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, these results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.

Zigman, M., Laumann-Lipp, N., Titus, T., Postlethwait, J. and Moens, C. B. (2014). Hoxb1b controls oriented cell division, cell shape and microtubule dynamics in neural tube morphogenesis. Development 141: 639-649. PubMed ID: 24449840
Summary: Hox genes are classically ascribed to function in patterning the anterior-posterior axis of bilaterian animals; however, their role in directing molecular mechanisms underlying morphogenesis at the cellular level remains largely unstudied. This study has unveil a non-classical role for the zebrafish hoxb1b gene (Drosophila homolog: Labial), which shares ancestral functions with mammalian Hoxa1, in controlling progenitor cell shape and oriented cell division during zebrafish anterior hindbrain neural tube morphogenesis. This is likely distinct from its role in cell fate acquisition and segment boundary formation. Without affecting major components of apico-basal or planar cell polarity, Hoxb1b regulates mitotic spindle rotation during the oriented neural keel symmetric mitoses that are required for normal neural tube lumen formation in the zebrafish. This function correlates with a non-cell-autonomous requirement for Hoxb1b in regulating microtubule plus-end dynamics in progenitor cells in interphase. It is proposed that Hox genes can influence global tissue morphogenesis by control of microtubule dynamics in individual cells in vivo.

Williams, D. R., Shifley, E. T., Lather, J. D. and Cole, S. E. (2014). Posterior skeletal development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis. Dev Biol 388: 159-169. PubMed ID: 24560643
Summary: The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng; see Drosophila Fringe) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. Mice lacking overt oscillatory Lfng expression in the posterior PSM (LfngFCE) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, an allelic series was created that progressively lowers Lfng levels in the PSM. Interestingly, it was found that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, while low levels of oscillatory Lfng in the posterior PSM were found to be sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development.

Gerhardt, D. M., et al. (2014). The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells. Genes Dev 28: 576-593. PubMed ID: 24637115
Summary: Notch1 (see Drosophila Notch) is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. This study used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj (see Drosophila Suppressor of Hairless), a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.

Saturday, March 22nd

Ishiguro, K., Kim, J., Shibuya, H., Hernandez-Hernandez, A., Suzuki, A., Fukagawa, T., Shioi, G., Kiyonari, H., Li, X. C., Schimenti, J., Hoog, C. and Watanabe, Y. (2014). Meiosis-specific cohesin mediates homolog recognition in mouse spermatocytes. Genes Dev 28: 594-607. PubMed ID: 24589552
Summary: During meiosis, homologous chromosome (homolog) pairing is promoted by several layers of regulation that include dynamic chromosome movement and meiotic recombination. However, the way in which homologs recognize each other remains a fundamental issue in chromosome biology. This study shows that homolog recognition or association initiates upon entry into meiotic prophase before axis assembly and double-strand break (DSB) formation. This homolog association develops into tight pairing only during or after axis formation. Intriguingly, the ability to recognize homologs is retained in Sun1 knockout spermatocytes, in which telomere-directed chromosome movement is abolished, and this is the case even in Spo11 knockout spermatocytes, in which DSB-dependent DNA homology search is absent. Disruption of meiosis-specific cohesin RAD21L (see Drosophila Rad/21) precludes the initial association of homologs as well as the subsequent pairing in spermatocytes. These findings suggest the intriguing possibility that homolog recognition is achieved primarily by searching for homology in the chromosome architecture as defined by meiosis-specific cohesin rather than in the DNA sequence itself.

Ballister, E. R., Riegman, M. and Lampson, M. A. (2014). Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint. J Cell Biol 204: 901-908. PubMed ID: 24637323
Summary: The mitotic checkpoint monitors kinetochore-microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, this study shows that recruiting the checkpoint protein Mad1 (see Drosophila Mad1) to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 (see Drosophila Altered disjunction) or BubR1. Furthermore, Mad2 (see Drosophila Mad2) binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of this checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores.

Moyle, M. W., Kim, T., Hattersley, N., Espeut, J., Cheerambathur, D. K., Oegema, K. and Desai, A. (2014). A Bub1-Mad1 interaction targets the Mad1-Mad2 complex to unattached kinetochores to initiate the spindle checkpoint. J Cell Biol 204: 647-657. PubMed ID: 24567362
Summary: Recruitment of Mad1-Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1-Mad2 to kinetochores is unclear. This paper shows that MAD-1 (see Drosophila Mad1) interacts with BUB-1 (see Drosophila Bud1) in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacts with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevent MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1-MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling.

Nabti, I., Marangos, P., Bormann, J., Kudo, N. R. and Carroll, J. (2014). Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity. J Cell Biol 204: 891-900. PubMed ID: 24637322
Summary: Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1; see Drosophila Cdc2) and mitogen-activated protein kinase (MAPK; see Drosophila Rolled). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. This study finds that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes.

Friday, March 21st

Holt, J. E., Pye, V., Boon, E., Stewart, J. L., Garcia-Higuera, I., Moreno, S., Rodriguez, R., Jones, K. T. and McLaughlin, E. A. (2014). The APC/C activator FZR1 is essential for meiotic prophase I in mice. Development 141: 1354-1365. PubMed ID: 24553289
Summary: Fizzy-related 1 (FZR1: see Drosophila Fizzy related) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model this study examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Delta mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. It is concluded that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.

Hehnly, H. and Doxsey, S. (2014). Rab11 endosomes contribute to mitotic spindle organization and orientation. Dev Cell 28: 497-507. PubMed ID: 24561039
Summary: During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 (see Drosophila Rab-protein 11) endosomes in mitosis. This paper show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. Mitotic recycling endosomes were found to bind γ-TuRC (see Drosophila γTubulin at 23C) components and associate with tubulin in vitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. It is proposes that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Bade, D., Pauleau, A. L., Wendler, A. and Erhardt, S. (2014). The E3 Ligase CUL3/RDX Controls Centromere Maintenance by Ubiquitylating and Stabilizing CENP-A in a CAL1-Dependent Manner. Dev Cell 28: 508-519. PubMed ID: 24636256
Summary: Centromeres are defined by the presence of the histone H3 variant centromere identifier (CENP-A) in a subset of centromeric nucleosomes. CENP-A deposition to centromeres depends on a specialized loading factor from yeast to humans that is called chromosome alignment defect 1 (CAL1) in Drosophila. This study shows that CAL1 directly interacts with roadkill (RDX), an adaptor for CUL3-mediated ubiquitylation. However, CAL1 is not a substrate of the CUL3/RDX ligase but functions as an additional substrate-specifying factor for the CUL3/RDX-mediated ubiquitylation of CENP-A. Remarkably, ubiquitylation of CENP-A by CUL3/RDX does not trigger its degradation but stabilizes CENP-A and CAL1. Loss of RDX leads to a rapid degradation of CAL1 and CENP-A and to massive chromosome segregation defects during development. Essentially, this study identified a proteolysis-independent role of ubiquitin conjugation in centromere regulation that is essential for the maintenance of the centromere-defining protein CENP-A and its loading factor CAL1.

Parisi, F., Stefanatos, R. K., Strathdee, K., Yu, Y. and Vidal, M. (2014). Transformed Epithelia Trigger Non-Tissue-Autonomous Tumor Suppressor Response by Adipocytes via Activation of Toll and Eiger/TNF Signaling. Cell Rep 6(5): 855-67. PubMed ID: 24582964
Summary: High tumor burden is associated with increased levels of circulating inflammatory cytokines that influence the pathophysiology of the tumor and its environment. The cellular and molecular events mediating the organismal response to a growing tumor are poorly understood. This paper reports a bidirectional crosstalk between epithelial tumors and the fat body - a peripheral immune tissue - in Drosophila. Tumors trigger a systemic immune response through activation of Eiger/TNF signaling, which leads to Toll pathway upregulation in adipocytes. Reciprocally, Toll elicits a non-tissue-autonomous program in adipocytes, which drives tumor cell death. Hemocytes play a critical role in this system by producing the ligands Spatzle and Eiger, which are required for Toll activation in the fat body and tumor cell death. Altogether, these results provide a paradigm for a long-range tumor suppression function of adipocytes in Drosophila, which may represent an evolutionarily conserved mechanism in the organismal response to solid tumors.

Thursday, March 20th

Akiyama, R., Masuda, M., Tsuge, S., Bessho, Y. and Matsui, T. (2014). An anterior limit of FGF/Erk signal activity marks the earliest future somite boundary in zebrafish. Development 141: 1104-1109. PubMed ID: 24504340
Summary: Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. This study characterized fibroblast growth factor (FGF; see Drosophila Branchless) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk (see Drosophila Rolled) activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. These results provide the first molecular evidence of the future somite boundary at B-5, and it is proposed that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development.

Lebreton, G. and Casanova, J. (2014). Specification of leading and trailing cell features during collective migration in the Drosophila trachea. J Cell Sci 127: 465-474. PubMed ID: 24213534
Summary: The role of tip and rear cells in collective migration is still a matter of debate and their differences at the cytoskeletal level are poorly understood. This study analysed these issues in the Drosophila trachea, an organ that develops from the collective migration of clusters of cells that respond to Branchless (Bnl), a fibroblast growth factor (FGF) homologue expressed in surrounding tissues. Individual cells in the migratory cluster were tracked and their features were characterized, and two prototypical types of cytoskeletal organisation were unveiled that account for tip and rear cells respectively. Indeed, once the former are specified, they remain as such throughout migration. Furthermore, FGF signalling in a single tip cell could trigger the migration of the cells in the branch. Finally, specific Rac activation was found at the tip cells, and a how FGF-independent cell features, such as adhesion and motility, act on coupling the behaviour of trailing and tip cells, was analyzed. Thus, the combined effect of FGF promoting leading cell behaviour and the modulation of cell properties in a cluster can account for the wide range of migratory events driven by FGF.

Mao, C. X., Xiong, Y., Xiong, Z., Wang, Q., Zhang, Y. Q. and Jin, S. (2014). Microtubule-severing protein Katanin regulates neuromuscular junction development and dendritic elaboration in Drosophila. Development 141: 1064-1074. PubMed ID: 24550114
Summary: Microtubules (MTs) are crucial for diverse biological processes including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. MT abnormalities are associated with neurodevelopmental and neurodegenerative diseases such as hereditary spastic paraplegia. Among many MT regulators, katanin was the first identified MT-severing protein, but its neuronal functions have not yet been examined in a multicellular organism. Katanin consists of two subunits; the catalytic subunit Katanin 60 contains an AAA (ATPases associated with a variety of cellular activities) domain and breaks MT fibers while hydrolyzing ATP, whereas Katanin 80 is a targeting and regulatory subunit. To dissect the in vivo functions of Katanin, Mutations were generated in Drosophila Katanin 60, and its expression was manipulated in a tissue-specific manner. Null mutants of Katanin 60 are pupal lethal, demonstrating that it is essential for viability. Loss-of-function mutants of Katanin 60 showed excess satellite boutons, reduced neurotransmission efficacy, and more enlarged cisternae at neuromuscular junctions. In peripheral sensory neurons, loss of Katanin 60 led to increased elaboration of dendrites, whereas overexpression of Katanin 60 resulted in the opposite. Genetic interaction analyses indicated that increased levels of MT acetylation increase its susceptibility to Katanin-mediated severing in neuronal and non-neuronal systems. Taken together, these results demonstrate for the first time that Katanin 60 is required for the normal development of neuromuscular synapses and dendrites.

Moffat, L. L., Robinson, R. E., Bakoulis, A. and Clark, S. G. (2014). The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans. Development 141: 617-628 PubMed ID: 24401370
Summary: Wnts (see Drosophila Wingless) control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. This study identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors (see Drosophila Frizzled) in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror (see Drosophila Ror) and LIN-18/Ryk (see Drosophila Derailed). PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. It is proposed that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development.

Wednesday, March 19th

Tie, F., Banerjee, R., Saiakhova, A. R., Howard, B., Monteith, K. E., Scacheri, P. C., Cosgrove, M. S. and Harte, P. J. (2014). Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing. Development 141: 1129-1139. PubMed ID: 24550119
Summary: Drosophila Trithorax (TRX) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these TRX functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. This study reports that the SET domains of TRX and TRX-related (TRR) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of TRX and Tyr2404 of TRR prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by TRX-dependent H3K27 acetylation by CREB-binding protein (CBP). Perturbation of Polycomb silencing by TRX overexpression was shown to require CBP. TRX and TRR are each physically associated with CBP in vivo, TRX binds directly to the CBP KIX domain, and the chromatin binding patterns of TRX and TRR are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of TRX and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory.

Cai, W., Wang, C., Li, Y., Yao, C., Shen, L., Liu, S., Bao, X., Schnable, P. S., Girton, J., Johansen, J. and Johansen, K. M. (2014). Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 24598257
Summary: This study determined the genome-wide relationship of Drosophila JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. In wild-type salivary gland cells the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing.

Yi, S. H., He, X. B., Rhee, Y. H., Park, C. H., Takizawa, T., Nakashima, K. and Lee, S. H. (2014). Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation. Development 141: 761-772. PubMed ID: 24496614
Summary: Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. This study demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 (Drosophila homolog Hormone receptor-like in 38) and Foxa2 (see Drosophila Forkhead). Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST (see Drosophila CoRest), and then recruiting histone deacetylase 1 (Hdac1; Drosophila homolog, Rpd3), an enzyme catalyzing histone deacetylation, to DA gene promoters. Co-expression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoREST-Hdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development.

Tee, W. W., Shen, S. S., Oksuz, O., Narendra, V. and Reinberg, D. (2014). Erk1/2 Activity Promotes Chromatin Features and RNAPII Phosphorylation at Developmental Promoters in Mouse ESCs. Cell 156: 678-690. PubMed ID: 24529373
Summary: Erk1/2 (see Drosophila Rolled) activation contributes to mouse ES cell pluripotency. This study found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 (see Drosophila Jumonji, AT rich interactive domain 2) and PRC2 (see Drosophila PRC2). Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII (see Drosophila RNA polymerase II) at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation.

Tuesday, March 18th

Laumonnerie, C., Da Silva, R. V., Kania, A. and Wilson, S. I. (2014). Netrin 1 and Dcc signalling are required for confinement of central axons within the central nervous system. Development 141: 594-603. PubMed ID: 24449837
Summary: The establishment of anatomically stereotyped axonal projections is fundamental to neuronal function. While most neurons project their axons within the central nervous system (CNS), only axons of centrally born motoneurons and peripherally born sensory neurons link the CNS and peripheral nervous system (PNS) together by navigating through specialized CNS/PNS transition zones. Such selective restriction is of importance because inappropriate CNS axonal exit could lead to loss of correct connectivity and also to gain of erroneous functions. However, to date, surprisingly little is known about the molecular-genetic mechanisms that regulate how central axons are confined within the CNS during development. This study shows that netrin 1/Dcc/Unc5 chemotropism contributes to axonal confinement within the CNS. In both Ntn1 (see Drosophila Netrins) and Dcc (see Drosophila Frazzled) mutant mouse embryos, some spinal interneuronal axons exit the CNS by traversing the CNS/PNS transition zones normally reserved for motor and sensory axons. Evidence that netrin 1 signalling preserves CNS/PNS axonal integrity in three ways: (1) netrin 1/Dcc ventral attraction diverts axons away from potential exit points; (2) a Dcc/Unc5c-dependent netrin 1 chemoinhibitory barrier in the dorsolateral spinal cord prevents interneurons from being close to the dorsal CNS/PNS transition zone; and (3) a netrin 1/Dcc-dependent, Unc5c-independent mechanism that actively prevents exit from the CNS. Together, these findings provide insights into the molecular mechanisms that maintain CNS/PNS integrity and present the first evidence that chemotropic signalling regulates interneuronal CNS axonal confinement in vertebrates.

Inamata, Y. and Shirasaki, R. (2014). Dbx1 triggers crucial molecular programs required for midline crossing by midbrain commissural axons. Development 141: 1260-1271. PubMed ID: 24553291
Summary: Axon guidance by commissural neurons has been well documented, providing a molecular logic of how midline crossing is achieved during development. Despite these advances, knowledge of the intrinsic genetic programs is still limited and it remains obscure whether the expression of a single transcription factor is sufficient to activate transcriptional programs that ultimately enable midline crossing. This study shows in the mouse that the homeodomain transcription factor Dbx1 (see Drosophila Dbx) is expressed by a subset of progenitor cells that give rise to commissural neurons in the dorsal midbrain. Gain- and loss-of-function analyses indicate that the expression of Dbx1 alone is sufficient and necessary to trigger midline crossing in vivo. It was also shown that Robo3 (see Drosophila Roundabout) controls midline crossing as a crucial downstream effector of the Dbx1-activated molecular programs. Furthermore, Dbx1 suppresses the expression of the transcriptional program for ipsilateral neuron differentiation in parallel. These results suggest that a single transcription factor, Dbx1, has an essential function in assigning midline-crossing identity, thereby contributing crucially to the establishment of the wiring laterality in the developing nervous system.

Oustah, A. A., Danesin, C., Khouri-Farah, N., Farreny, M. A., Escalas, N., Cochard, P., Glise, B. and Soula, C. (2014). Dynamics of Sonic hedgehog signaling in the ventral spinal cord are controlled by intrinsic changes in source cells requiring Sulfatase 1. Development 141: 1392-1403. PubMed ID: 24595292
Summary: In the ventral spinal cord, generation of neuronal and glial cell subtypes is controlled by Sonic hedgehog (Shh; see Drosophila Hedgehog). This morphogen contributes to cell diversity by regulating spatial and temporal sequences of gene expression during development. This study reports that establishing Shh source cells is not sufficient to induce the high-threshold response required to specify sequential generation of ventral interneurons and oligodendroglial cells at the right time and place in zebrafish. Instead, it was shown that Shh-producing cells must repeatedly upregulate the secreted enzyme Sulfatase1 (Sulf1) at two critical time points of development to reach their full inductive capacity. Evidence is provided that Sulf1 triggers Shh signaling activity to establish and, later on, modify the spatial arrangement of gene expression in ventral neural progenitors. Arguments are further presented in favor of Sulf1 controlling Shh temporal activity by stimulating production of active forms of Shh from its source. This work, by pointing out the key role of Sulf1 in regulating Shh-dependent neural cell diversity, highlights a novel level of regulation, which involves temporal evolution of Shh source properties.

Klimova, L. and Kozmik, Z. (2014). Stage-dependent requirement of neuroretinal Pax6 for lens and retina development. Development 141: 1292-1302. PubMed ID: 24523460
Summary: The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 (see Drosophila Eyeless) in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. This study shows that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6(Sey/flox) embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. These data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner.

Monday, March 17th

Janssens, D. H., Komori, H., Grbac, D., Chen, K., Koe, C. T., Wang, H. and Lee, C. Y. (2014). Earmuff restricts progenitor cell potential by attenuating the competence to respond to self-renewal factors. Development 141: 1036-1046. PubMed ID: 24550111
Summary: Despite expressing stem cell self-renewal factors, intermediate progenitor cells possess restricted developmental potential, which allows them to give rise exclusively to differentiated progeny rather than stem cell progeny. Failure to restrict the developmental potential can allow intermediate progenitor cells to revert into aberrant stem cells that might contribute to tumorigenesis. Insight into stable restriction of the developmental potential in intermediate progenitor cells could improve understanding of the development and growth of tumors, but the mechanisms involved remain largely unknown. Intermediate neural progenitors (INPs), generated by type II neural stem cells (neuroblasts) in fly larval brains, provide an in vivo model for investigating the mechanisms that stably restrict the developmental potential of intermediate progenitor cells. This study reports that the transcriptional repressor protein Earmuff (Erm) functions temporally after Brain tumor (Brat) and Numb to restrict the developmental potential of uncommitted (immature) INPs. Consistently, endogenous Erm is detected in immature INPs but undetectable in INPs. Erm-dependent restriction of the developmental potential in immature INPs tleads to attenuated competence to respond to all known neuroblast self-renewal factors in INPs. he BAP chromatin-remodeling complex probably functions cooperatively with Erm to restrict the developmental potential of immature INPs. Together, these data have led to conclude that the Erm-BAP-dependent mechanism stably restricts the developmental potential of immature INPs by attenuating their genomic responses to stem cell self-renewal factors. It is proposed that restriction of developmental potential by the Erm-BAP-dependent mechanism functionally distinguishes intermediate progenitor cells from stem cells, ensuring the generation of differentiated cells and preventing the formation of progenitor cell-derived tumor-initiating stem cells.

Lyons, G. R., Andersen, R. O., Abdi, K., Song, W. S. and Kuo, C. T. (2014). Cysteine Proteinase-1 and Cut Protein Isoform Control Dendritic Innervation of Two Distinct Sensory Fields by a Single Neuron. Cell Rep [Epub ahead of print]. PubMed ID: 24582961
Summary: Dendrites often exhibit structural changes in response to local inputs. Although mechanisms that pattern and maintain dendritic arbors are becoming clearer, processes regulating regrowth, during context-dependent plasticity or after injury, remain poorly understood. This study found that a class of Drosophila sensory neurons, through complete pruning and regeneration, can elaborate two distinct dendritic trees, innervating independent sensory fields. An expression screen identified Cysteine proteinase-1 (Cp1) as a critical regulator of this process. Unlike known ecdysone effectors, Cp1-mutant ddaC neurons pruned larval dendrites normally but fail to regrow adult dendrites. Cp1 expression is upregulated/concentrated in the nucleus during metamorphosis, controlling production of a truncated Cut homeodomain transcription factor. This truncated Cut, but not the full-length protein, allows Cp1-mutant ddaC neurons to regenerate higher-order adult dendrites. These results identify a molecular pathway needed for dendrite regrowth after pruning, which allows the same neuron to innervate distinct sensory fields.

Xu, D., Zhang, F., Wang, Y., Sun, Y. and Xu, Z. (2014). Microcephaly-associated protein WDR62 regulates neurogenesis through JNK1 in the developing neocortex. Cell Rep 6: 104-116. PubMed ID: 24388750
Summary: Mutations of WD40-repeat protein 62 (WDR62) have been identified recently to cause human MCPH (autosomal-recessive primary microcephaly), a neurodevelopmental disorder characterized by decreased brain size. However, the underlying mechanism is unclear. This study investigated the function of WDR62 in brain development and the pathological role of WDR62 mutations. WDR62 knockdown was found to lead to premature differentiation of neural progenitor cells (NPCs). The defect can be rescued by wild-type human WDR62, but not by the five MCPH-associated WDR62 mutants. It was demonstrate that WDR62 acts upstream of JNK signaling in the control of neurogenesis. Depletion of JNK1 and WDR62 incurs very similar defects including abnormal spindle formation and mitotic division of NPCs as well as premature NPC differentiation during cortical development. Thus, these findings indicate that WDR62 is required for proper neurogenesis via JNK1 and provide an insight into the molecular mechanisms underlying MCPH pathogenesis.

Kim, J., Kwon, J. T., Kim, H. S., Josselyn, S. A. and Han, J. H. (2014). Memory recall and modifications by activating neurons with elevated CREB. Nat Neurosci 17: 65-72. PubMed ID: 24212670
Summary: Memory is supported by a specific ensemble of neurons distributed in the brain that form a unique memory trace. Previous studies have shown that neurons in the lateral amygdala expressing elevated levels of cAMP response-element binding protein (see Drosophila Cbp/Nejire) are preferentially recruited into fear memory traces and are necessary for the expression of those memories. However, it is unknown whether artificially activating just these selected neurons in the absence of behavioral cues is sufficient to recall that fear memory. Using an ectopic rat vanilloid receptor TRPV1 and capsaicin system, this study found that activating this specific ensemble of neurons was sufficient to recall established fear memory. Furthermore, this neuronal activation induced a reconsolidation-like reorganization process, or strengthening of the fear memory. Thus, these findings establish a direct link between the activation of specific ensemble of neurons in the lateral amygdala and the recall of fear memory and its subsequent modifications.

Sunday, March 16th

Zamudio, J. R., Kelly, T. J. and Sharp, P. A. (2014). Argonaute-Bound Small RNAs from Promoter-Proximal RNA Polymerase II. Cell 156: 920-934. PubMed ID: 24581493
Summary: Argonaute (Ago; see Drosophila RNAi and Posttranscriptional Gene Silencing) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, a mouse embryonic stem cell system was adapted to express a single epitope-tagged Ago protein family member in an inducible manner. This paper reports the small RNA profile of Ago-deficient cells and shows that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, an Ago-dependent class of noncanonical miRNAs was identified derived from protein-coding gene promoters, which were named transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, evidence is presented of differential expression and conservation in humans, suggesting important roles in gene regulation.

Mori, M., Triboulet, R., Mohseni, M., Schlegelmilch, K., Shrestha, K., Camargo, F. D. and Gregory, R. I. (2014). Hippo Signaling Regulates Microprocessor and Links Cell-Density-Dependent miRNA Biogenesis to Cancer. Cell 156: 893-906. PubMed ID: 24581491
Summary: Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. This study shows that YAP (see Drosophila Yorkie), the downstream target of the tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding posttranscriptional induction of MYC (see Drosophila Myc) expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.

Chawla, G. and Sokol, N. S. (2014). ADAR mediates differential expression of polycistronic microRNAs. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 24561617
Summary: Adenosine deaminases acting on RNAs (ADARs) convert adenosine residues to inosines in primary microRNA (pri-miRNA) transcripts to alter the structural conformation of these precursors and the subsequent functions of the encoded microRNAs (miRNAs). This study shows that RNA editing by Drosophila ADAR modulates the expression of three co-transcribed miRNAs encoded by the evolutionarily conserved let-7-Complex (let-7-C) locus. For example, a single A-to-I change at the -6 residue of pri-miR-100, the first miRNA in this let-7 polycistronic transcript, leads to enhanced miRNA processing by Drosha and consequently enhanced functional miR-100 both in vitro as well as in vivo. In contrast, other editing events, including one at the +43 residue of the pri-miR-125, destabilize the primary transcript and reduce the levels of all three encoded miRNAs. Consequently, loss of Adar in vivo leads to reduced miR-100 but increased miR-125. In wild-type animals, the destabilizing editing events in pri-let-7-C increase during the larval-to-adult transition and are critical for the normal downregulation of all three miRNAs seen late in metamorphosis. These findings unravel a new regulatory role for ADAR and raise the possibility that ADAR mediates the differential expression characteristic of many polycistronic miRNA clusters.

Jambor, H., Mueller, S., Bullock, S. L. and Ephrussi, A. (2014). A stem-loop structure directs oskar mRNA to microtubule minus ends. RNA [Epub ahead of print]. PubMed ID: 24572808
Summary: mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. This study shows that a 67-nt stem-loop in the oskar 3' UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. This work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.

Saturday, March 15th

Kim, T. H., Li, F., Ferreiro-Neira, I., Ho, L. L., Luyten, A., Nalapareddy, K., Long, H., Verzi, M. and Shivdasani, R. A. (2014). Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506: 511-515. PubMed ID: 24413398
Summary: Cells differentiate when transcription factors bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem cells, chromatin at lineage-restricted genes becomes sequentially accessible, probably by means of 'pioneer' transcription factor activity, but tissues may use other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbours, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. This study reports the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where notch signalling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages. Transcript profiles in isolated leucine-rich repeat-containing receptor 5 (LGR5) positive intestinal stem cells and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks (see Drosophila Histone 3) and DNase I hypersensitivity - signifying accessible, permissive chromatin - at most of the same cis-elements. Enhancers acting uniquely in progenitors were well demarcated in LGR5+ intestinal stem cells, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1 (see Drosophila Atonal), a secretory-specific transcription factor, controls lateral inhibition through delta-like notch ligand genes and also drives the expression of numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to the presence or absence of a key transcription factor. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted transcription factor with broadly permissive chromatin established in multipotent stem cells.

Xu, J., Liu, H., Park, J. S., Lan, Y. and Jiang, R. (2014). Osr1 acts downstream of and interacts synergistically with Six2 to maintain nephron progenitor cells during kidney organogenesis. Development [Epub ahead of print]. PubMed ID: 24598167
Summary: Mammalian kidney organogenesis involves reciprocal epithelial-mesenchymal interactions that drive iterative cycles of nephron formation. Recent studies have demonstrated that the Six2 transcription factor (see Drosophila Sine oculis) acts cell autonomously to maintain nephron progenitor cells, whereas canonical Wnt signaling induces nephron differentiation. How Six2 maintains the nephron progenitor cells against Wnt-directed commitment is not well understood, however. This study reports that Six2 is required to maintain expression of Osr1, a homolog of the Drosophila odd-skipped zinc-finger transcription factor, in the undifferentiated cap mesenchyme. Tissue-specific inactivation of Osr1 in the cap mesenchyme causes premature depletion of nephron progenitor cells and severe renal hypoplasia. Osr1 and Six2 act synergistically to prevent premature differentiation of the cap mesenchyme. Furthermore, although both Six2 and Osr1 could form protein interaction complexes with TCF proteins, Osr1, but not Six2, enhances TCF (see Drosophila Pangolin) interaction with the Groucho family transcriptional co-repressors (see Drosophila Groucho). Loss of Osr1 was shown to result in β-catenin/TCF-mediated ectopic activation of Wnt4 enhancer-driven reporter gene expression in the undifferentiated nephron progenitor cells in vivo. Together, these data indicate that Osr1 plays crucial roles in Six2-dependent maintenance of nephron progenitors during mammalian nephrogenesis by stabilizing TCF-Groucho transcriptional repressor complexes to antagonize Wnt-directed nephrogenic differentiation.

Jusiak, B., Karandikar, U. C., Kwak, S. J., Wang, F., Wang, H., Chen, R. and Mardon, G. (2014). Regulation of Drosophila Eye Development by the Transcription Factor Sine oculis. PLoS One 9: e89695. PubMed ID: 24586968
Summary: Homeodomain transcription factors of the Sine oculis (SIX) family direct multiple regulatory processes throughout the metazoans. Sine oculis (So) was first characterized in the fruit fly Drosophila melanogaster, where it is both necessary and sufficient for eye development, regulating cell survival, proliferation, and differentiation. Despite its key role in development, only a few direct targets of So have been described previously. The current study aimed to expand knowledge of So-mediated transcriptional regulation in the developing Drosophila eye using ChIP-seq to map So binding regions throughout the genome. Over seven thousand So enriched regions (peaks), estimated to map to over five thousand genes. Using overlap between the So ChIP-seq peak set and genes that are differentially regulated in response to loss or gain of so, putative direct targets of So were identified. So binding enrichment was found in genes not previously known to be regulated by So, including genes that encode cell junction proteins and signaling pathway components. In addition, a subset of So-bound novel genes were examined in the eye, and eight genes were found that have previously uncharacterized eye phenotypes and may be novel direct targets of So. Analysis of So target senseless (sens) suggested a three-level feed-forward loop, whereby Eyeless and So activate each other, So and Ey activate ato, and So and Ato activate sens. This study presents a greatly expanded list of candidate So targets and serves as basis for future studies of So-mediated gene regulation in the eye.

Delaunay, D., Cortay, V., Patti, D., Knoblauch, K. and Dehay, C. (2014). Mitotic Spindle Asymmetry: A Wnt/PCP-Regulated Mechanism Generating Asymmetrical Division in Cortical Precursors. Cell Rep 6: 400-414. PubMed ID:
Summary: The regulation of asymmetric cell division (ACD) during corticogenesis is incompletely understood. This study documents that spindle-size asymmetry (SSA) between the two poles occurs during corticogenesis and parallels ACD. SSA appears at metaphase and is maintained throughout division, and it is necessary for proper neurogenesis. Imaging of spindle behavior and division outcome reveals that neurons preferentially arise from the larger-spindle pole. Mechanistically, SSA magnitude is controlled by Wnt7a (see Drosophila Wingless) and Vangl2 (see Drosophila Van Gogh), both members of the Wnt/planar cell polarity (PCP)-signaling pathway, and relayed to the cell cortex by P-ERM proteins. In vivo, Vangl2 and P-ERM (see Drosophila Moesin) downregulation promotes early cell-cycle exit and prevents the proper generation of late-born neurons. Thus, SSA is a core component of ACD that is conserved in invertebrates and vertebrates and plays a key role in the tight spatiotemporal control of self-renewal and differentiation during mammalian corticogenesis.

Friday, March 14th

Roussel, E., Carcaud, J., Combe, M., Giurfa, M. and Sandoz, J. C. (2014). Olfactory coding in the honeybee lateral horn. Curr Biol 24: 561-567. PubMed ID: 24560579
Summary: Olfactory systems dynamically encode odor information in the nervous system. Insects constitute a well-established model for the study of the neural processes underlying olfactory perception. In insects, odors are detected by sensory neurons located in the antennae, whose axons project to a primary processing center, the antennal lobe. There, the olfactory message is reshaped and further conveyed to higher-order centers, the mushroom bodies and the lateral horn. Previous work has intensively analyzed the principles of olfactory processing in the antennal lobe and in the mushroom bodies. However, how the lateral horn participates in olfactory coding remains comparatively more enigmatic. This work studied odor representation at the input to the lateral horn of the honeybee, a social insect that relies on both floral odors for foraging and pheromones for social communication. Using in vivo calcium imaging, consistent neural activity was shown in the honeybee lateral horn upon stimulation with both floral volatiles and social pheromones. Recordings reveal odor-specific maps in this brain region as stimulations with the same odorant elicit more similar spatial activity patterns than stimulations with different odorants. Odor-similarity relationships are mostly conserved between antennal lobe and lateral horn, so that odor maps recorded in the lateral horn allow predicting bees' behavioral responses to floral odorants. In addition, a clear segregation of odorants based on pheromone type is found in both structures. The lateral horn thus contains an odor-specific map with distinct representations for the different bee pheromones, a prerequisite for eliciting specific behaviors.

Silva, B., Goles, N. I., Varas, R. and Campusano, J. M. (2014). Serotonin receptors expressed in Drosophila mushroom bodies differentially modulate larval locomotion. PLoS One 9: e89641. PubMed ID: 24586928
Summary: Drosophila melanogaster has been successfully used as a simple model to study the cellular and molecular mechanisms underlying behaviors, including the generation of motor programs. Thus, it has been shown that, as in vertebrates, CNS biogenic amines (BA) including serotonin (5HT) participate in motor control in Drosophila. BA systems innervate an important association area in the insect brain previously associated to the planning and/or execution of motor programs, the Mushroom Bodies (MB). The main objective of this work is to evaluate the contribution of 5HT and its receptors expressed in MB to motor behavior in fly larva. Locomotion was evaluated using an automated tracking system, in Drosophila larvae (3rd-instar) exposed to drugs that affect the serotonergic neuronal transmission: alpha-methyl-L-dopa, MDMA and fluoxetine. In addition, animals expressing mutations in the 5HT biosynthetic enzymes or in any of the previously identified receptors for this amine (5HT1AR, 5HT1BR, 5HT2R and 5HT7R) were evaluated in their locomotion. Finally, RNAi directed to the Drosophila 5HT receptor transcripts were expressed in MB and the effect of this manipulation on motor behavior was assessed. Data obtained in the mutants and in animals exposed to the serotonergic drugs, suggest that 5HT systems are important regulators of motor programs in fly larvae. Studies carried out in animals pan-neuronally expressing the RNAi for each of the serotonergic receptors, support this idea and further suggest that CNS 5HT pathways play a role in motor control. Moreover, animals expressing an RNAi for 5HT1BR, 5HT2R and 5HT7R in MB show increased motor behavior, while no effect is observed when the RNAi for 5HT1AR is expressed in this region. Thus, these data suggest that CNS 5HT systems are involved in motor control, and that 5HT receptors expressed in MB differentially modulate motor programs in fly larvae.

Cho, J. Y. and Sternberg, P. W. (2014). Multilevel modulation of a sensory motor circuit during C. elegans sleep and arousal. Cell 156: 249-260. PubMed ID: 24439380
Summary: Sleep is characterized by behavioral quiescence, homeostasis, increased arousal threshold, and rapid reversibility. Understanding how these properties are encoded by a neuronal circuit has been difficult, and no single molecular or neuronal pathway has been shown to be responsible for the regulation of sleep. Taking advantage of the well-mapped neuronal connections of Caenorhabditis elegans and the sleep-like states in this animal, this study demonstrated the changed properties of both sensory neurons and downstream interneurons that mediate sleep and arousal. The ASH sensory neuron displays reduced sensitivity to stimuli in the sleep-like state, and the activity of the corresponding interneurons in ASH's motor circuit becomes asynchronous. Restoration of interneuron synchrony is sufficient for arousal. The multilevel circuit depression revealed provides an elegant strategy to promote a robust decrease in arousal while allowing for rapid reversibility of the sleep state.

Bechstein, P., Rehbach, N. J., Yuhasingham, G., Schurmann, C., Gopfert, M., Kossl, M. and Maronde, E. (2014). The clock gene Period1 regulates innate routine behaviour in mice. Proc Biol Sci 281: 20140034. PubMed ID: 24598427
Summary: Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. This study found that in mice inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour.

Thursday, March 13th

Chen, B., Brinkmann, K., Chen, Z., Pak, C. W., Liao, Y., Shi, S., Henry, L., Grishin, N. V., Bogdan, S. and Rosen, M. K. (2014). The WAVE regulatory complex links diverse receptors to the actin cytoskeleton. Cell 156: 195-207. PubMed ID: 24439376
Summary: The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. This study has identified a large family of potential WRC ligands, consisting of approximately 120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sarah and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. These findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.

Soulavie, F., Piepenbrock, D., Thomas, J., Vieillard, J., Duteyrat, J. L., Cortier, E., Laurencon, A., Gopfert, M. C. and Durand, B. (2014). hemingway is required for sperm flagella assembly and ciliary motility in Drosophila. Mol Biol Cell [Epub ahead of print]. PubMed ID: 24554765
Summary: Cilia play major functions in physiology and development and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to Primary Ciliary Dyskinesia with upper airways recurrent infections, left-right asymmetry perturbations and fertility defects. This study has identified in Drosophila, hemingway (hmw, CG7669), as a novel component required for motile cilia function. hmw encodes a 604-amino-acid protein characterized by a highly conserved coiled-coil domain also found in the human ortholog KIAA1430. HMW is conserved in species with motile cilia and, in Drosophila, hmw is expressed in ciliated sensory neurons and in spermatozoa. hmw knock-out flies are hearing-impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, it is required for the elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, it is proposed that HMW is not a structural component of the motile axoneme, but is required for proper acquisition of motile properties. This identifies HMW as a novel evolutionarily conserved component necessary for motile cilium function and flagella assembly.

Bilancia, C. G., Winkelman, J. D., Tsygankov, D., Nowotarski, S. H., Sees, J. A., Comber, K., Evans, I., Lakhani, V., Wood, W., Elston, T. C., Kovar, D. R. and Peifer, M. (2014). Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivo. Dev Cell 28: 394-408. PubMed ID: 24576424
Summary: Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, it is important to address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. Diaphanous (Dia) and Enabled (Ena) were characterized as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. Dia and Ena were found to have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.

Winkelman, J. D., Bilancia, C. G., Peifer, M. and Kovar, D. R. (2014). Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24591594
Summary: Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin (Drosophila Singed). However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. This study used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of approximately 10 s in solution, for approximately 60 s when immobilized on a surface, and for approximately 110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, it was found that Ena's actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.

Wednesday, March 12th

Kunnapuu, J., Tauscher, P., Tiusanen, N., Nguyen, M., Loytynoja, A., Arora, K. and Shimmi, O. (2014). Cleavage of the Drosophila Screw prodomain is critical for a dynamic BMP morphogen gradient in embryogenesis. Dev Biol [Epub ahead of print]. PubMed ID: 24560644
Summary: Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, Decapentaplegic (Dpp) a BMP2/4 ortholog and Screw (Scw) a BMP5/6/7/8 family member. scwE1 encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. This study shows that scwE1 contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, the data show that ScwE1 preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scwE1 alleles. The unprocessed prodomain of ScwE1 remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.

Henstridge, M. A., Johnson, T. K., Warr, C. G. and Whisstock, J. C. (2014). Trunk cleavage is essential for Drosophila terminal patterning and can occur independently of Torso-like. Nat Commun 5: 3419. PubMed ID: 24584029
Summary: Terminal patterning in Drosophila is governed by a localized interaction between the Torso kinase (Tor) and its ligand Trunk (Trk). Currently, it is proposed that Trk must be cleaved in order to bind Tor, and that these proteolytic events are controlled by secretion of Torso-like (Tsl) only at the embryo poles. However, controversy surrounds these ideas since neither cleaved Trk nor a protease that functions in terminal patterning have been identified. This study shows that Trk is cleaved multiple times in vivo and that these proteolytic events are essential for its function. Unexpectedly, however, the Trk cleavage patterns are unaltered in tsl-null mutants. One explanation for these data is that the influence of Tsl on localized Trk cleavage at the embryo poles is subtle and cannot be readily detected. Alternatively, a scenario is favoured where Tsl functions post proteolytic processing of Trk to control localized terminal patterning.

He, B., Doubrovinski, K., Polyakov, O. and Wieschaus, E. (2014). Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation. Nature [Epub ahead of print]. PubMed ID: 24590071
Summary: Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is used throughout development in most animals. Little is known, however, about how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. This study applied particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow formation. It was found that cytoplasmic redistribution during the lengthening phase of ventral furrow formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to, or driving force on, the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells before gastrulation ('acellular' embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild-type embryos. These results indicate that during the lengthening phase of ventral furrow formation, hydrodynamic behaviour of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable.

Takayama, S., Dhahbi, J., Roberts, A., Mao, G., Heo, S. J., Pachter, L., Martin, D. and Boffelli, D. (2014). Genome methylation in D. melanogaster is found at specific short motifs and is independent of DNMT2 activity. Genome Res. PubMed ID: 24558263
Summary: Cytosine methylation in the genome of Drosophila has been elusive and controversial: its location and function have not been established. This study used a novel and highly sensitive genome-wide cytosine methylation assay to detect and map genome methylation in Stage 5 Drosophila embryos. The methylation observed with this method is highly localized and strand-asymmetrical, limited to regions covering ~1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. Gene body methylation is associated with lower expression, and many genes containing methylated regions have developmental or transcriptional functions. The only known DNA methyltransferase in Drosophila is DNMT2, but lines deficient for DNMT2 retain genomic methylation, implying the presence of a novel methyltransferase. The association of methylation with lower expression of specific developmental genes at Stage 5 raises the possibility that it participates in controlling gene expression during the maternal-zygotic transition (Takayama, 2014).

Tuesday, March 11th

Byrne, A. B., Walradt, T., Gardner, K. E., Hubbert, A., Reinke, V. and Hammarlund, M. (2014). Insulin/IGF1 Signaling Inhibits Age-Dependent Axon Regeneration. Neuron 81: 561-573. PubMed ID: 24440228
Summary: The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. This study shows that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2 (see Drosophila Insulin-like receptor). DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO . DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, DAF-18/PTEN (see Drosophila Pten) was found to inhibit regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1 (see Drosophila Wallenda), a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, these data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR.

Alic, N., Tullet, J. M., Niccoli, T., Broughton, S., Hoddinott, M. P., Slack, C., Gems, D. and Partridge, L. (2014). Cell-Nonautonomous Effects of dFOXO/DAF-16 in Aging. Cell Rep 6: 608-616. PubMed ID: 24508462
Summary: Drosophila melanogaster and Caenorhabditis elegans each carry a single representative of the Forkhead box O (FoxO) family of transcription factors, dFOXO and DAF-16, respectively. Both are required for lifespan extension by reduced insulin/Igf signaling, and their activation in key tissues can extend lifespan. Aging of these tissues may limit lifespan. Alternatively, FoxOs may promote longevity cell nonautonomously by signaling to themselves (FoxO to FoxO) or other factors (FoxO to other) in distal tissues. This study shows that activation of dFOXO and DAF-16 in the gut/fat body does not require dfoxo/daf-16 elsewhere to extend lifespan. Rather, in Drosophila, activation of dFOXO in the gut/fat body or in neuroendocrine cells acts on other organs to promote healthy aging by signaling to other, as-yet-unidentified factors. Whereas FoxO-to-FoxO signaling appears to be required for metabolic homeostasis, the current results pinpoint FoxO-to-other signaling as an important mechanism through which localized FoxO activity ameliorates aging.

Cao, J., Ni, J., Ma, W., Shiu, V., Milla, L. A., Park, S., Spletter, M. L., Tang, S., Zhang, J., Wei, X., Kim, S. K. and Scott, M. P. (2014). Insight into Insulin Secretion from Transcriptome and Genetic Analysis of Insulin-Producing Cells of Drosophila. Genetics [Epub ahead of print]. PubMed ID: 24558258
Summary: Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). Genomic and candidate gene approaches were used to identify components functioning in IPCs to control ILP production,. Laser microdissection and mRNA sequencing were used to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing beta cells of the mammalian pancreas. The genes in common encode insulin-like peptides and proteins that control insulin metabolism, storage, secretion, and beta cell proliferation, and some not previously linked to insulin production or beta cell function. Among these novelties is unc-104, a Kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, dominant-negative Rab genes were tested to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.

Okumura, T., Takeda, K., Taniguchi, K. and Adachi-Yamada, T. (2014). βnu Integrin Inhibits Chronic and High Level Activation of JNK to Repress Senescence Phenotypes in Drosophila Adult Midgut. PLoS One 9: e89387. PubMed ID: 24586740
Summary: Proper control of adult stem cells including their proliferation and differentiation is crucial in maintaining homeostasis of well-organized tissues/organs throughout an organism's life. The Drosophila adult midgut has intestinal stem cells (ISCs), which have been exploited as a simple model system to investigate mechanisms controlling adult tissue homeostasis. This study found that a viable mutant of βnu integrin (βint-nu), encoding one of two Drosophila integrin β subunits, showed a short midgut and abnormal multilayered epithelia accompanied by an increase in ISC proliferation and misdifferentiation defects. The increase in ISC proliferation and misdifferentiation was due to frequent ISC duplication expanding a pool of ISCs, which was caused by depression of the Notch signalling, and up-regulation of unpaired (upd), a gene encoding an extracellular ligand in the JAK/STAT signalling pathway. In addition, abnormally high accumulation of filamentous actin (F-actin) occurred in the βint-nu mutant enterocytes. Furthermore, the defects were rescued by suppressing c-Jun N-terminal kinase (JNK) signalling, which was up-regulated in a manner correlated with the defect levels in the above-mentioned βint-nu mutant phenotype. These symptoms observed in young βint-nu mutant midgut were very similar to those in the aged midgut in wild type. These results suggested that βint-nu has a novel function for the Drosophila adult midgut homeostasis under normal conditions and provided a new insight into possible age-related diseases caused by latent abnormality of an integrin function.

Monday, March 10th

Titen, S. W., Lin, H. C., Bhandari, J. and Golic, K. G. (2014). Chk2 and p53 regulate the transmission of healed chromosomes in the Drosophila male germline. PLoS Genet 10: e1004130. PubMed ID: 24586185
Summary: When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. This study found that Chk2 (encoded by loki) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. It is concluded that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.

Bolcun-Filas, E., Rinaldi, V. D., White, M. E. and Schimenti, J. C. (2014). Reversal of female infertility by Chk2 ablation reveals the oocyte DNA damage checkpoint pathway. Science 343: 533-536. PubMed ID: 24482479
Summary: Genetic errors in meiosis can lead to birth defects and spontaneous abortions. Checkpoint mechanisms of hitherto unknown nature eliminate oocytes with unrepaired DNA damage, causing recombination-defective mutant mice to be sterile. This study reports that checkpoint kinase 2 (Chk2 or Chek2: loki in Drosophila), is essential for culling mouse oocytes bearing unrepaired meiotic or induced DNA double-strand breaks (DSBs). Female infertility caused by a meiotic recombination mutation or irradiation was reversed by mutation of Chk2. Both meiotically programmed and induced DSBs trigger CHK2-dependent activation of TRP53 (p53) and TRP63 (p63), effecting oocyte elimination. These data establish CHK2 as essential for DNA damage surveillance in female meiosis and indicate that the oocyte DSB damage response primarily involves a pathway hierarchy in which ataxia telangiectasia and Rad3-related (ATR) signals to CHK2, which then activates p53 and p63.

Blachon, S., Khire, A. and Avidor-Reiss, T. (2014). The Origin of the Second Centriole in the Zygote of Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 24532732
Summary: Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila spermatids contain a single centriole called the giant centriole and a proximal centriole-like (PCL) structure whose function is unknown. This study found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins, and are seen in asterless mutant embryos that cannot form centrioles. The zygote's centriolar proteins label only the daughter centrioles of first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote, and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization.

Yuan, K., Shermoen, A. W. and O'Farrell, P. H. (2014). Illuminating DNA replication during Drosophila development using TALE-lights. Curr Biol 24: R144-145. PubMed ID: 24556431
Summary: New discoveries allow systematic engineering of DNA sequence recognition using the modular recognition units of the transcription activator-like effectors (TALEs) or the guide RNA of the CRISPRs. The engineered specificity offers the potential to guide a wide range of activities to particular sequences, and targeted nucleases cause directed mutagenesis. This study tagged sequences using fluorescent fusions that were called TALE-lights, and used these to follow replication of particular satellite sequences during a major embryonic transition in Drosophila. This study showed that replication-timing of individual sequences can be measured. As embryos develop, the cell cycle extends and the timing of replication of satellite sequences shifts to later times within the more prolonged S phase. The compact foci of satellite sequences expand in conjunction with their replication and a satellite sequence, 359-bp, shows a particularly marked shift to later replication in the cell cycle after the midblastula transition (MBT). This replication behavior suggests that developmental signals can separately influence the timing of different satellite sequences.

Sunday, March 9th

Otsuna, H., Shinomiya, K. and Ito, K. (2014). Parallel neural pathways in higher visual centers of the Drosophila brain that mediate wavelength-specific behavior. Front Neural Circuits 8: 8. PubMed ID: 24574974
Summary: Compared with connections between the retinae and primary visual centers, relatively less is known in both mammals and insects about the functional segregation of neural pathways connecting primary and higher centers of the visual processing cascade. Using the Drosophila visual system as a model, this study demonstrates two levels of parallel computation in the pathways that connect primary visual centers of the optic lobe to computational circuits embedded within deeper centers in the central brain. A seemingly simple achromatic behavior, namely phototaxis, is shown to be under the control of several independent pathways, each of which is responsible for navigation towards unique wavelengths. Silencing just one pathway is enough to disturb phototaxis towards one characteristic monochromatic source, whereas phototactic behavior towards white light is not affected. The response spectrum of each demonstrable pathway is different from that of individual photoreceptors, suggesting subtractive computations. A choice assay between two colors showed that these pathways are responsible for navigation towards, but not for the detection itself of, the monochromatic light. The present study provides novel insights about how visual information is separated and processed in parallel to achieve robust control of an innate behavior.

Hollis, B. and Kawecki, T. J. (2014). Male cognitive performance declines in the absence of sexual selection. Proc Biol Sci 281: 20132873. PubMed ID: 24573848
Summary: Sexual selection is responsible for the evolution of male ornaments and armaments, but its role in the evolution of cognition-the ability to process, retain and use information-is largely unexplored. Because successful courtship is likely to involve processing information in complex, competitive sexual environments, it is hypothesized that sexual selection contributes to the evolution and maintenance of cognitive abilities in males. To test this, mate choice and mate competition were removed from experimental populations of Drosophila melanogaster by enforcing monogamy for over 100 generations. Males evolved under monogamy became less proficient than polygamous control males at relatively complex cognitive tasks. When faced with one receptive and several unreceptive females, polygamous males quickly focused on receptive females, whereas monogamous males continued to direct substantial courtship effort towards unreceptive females. As a result, monogamous males were less successful in this complex setting, despite being as quick to mate as their polygamous counterparts with only one receptive female. This diminished ability to use past information was not limited to the courtship context: monogamous males (but not females) also showed reduced aversive olfactory learning ability. These results provide direct experimental evidence that the intensity of sexual selection is an important factor in the evolution of male cognitive ability.

Coen, P., Clemens, J., Weinstein, A. J., Pacheco, D. A., Deng, Y. and Murthy, M. (2014). Dynamic sensory cues shape song structure in Drosophila. Nature [Epub ahead of print]. PubMed ID: 24598544
Summary: The generation of acoustic communication signals is widespread across the animal kingdom, and males of many species, including Drosophilidae, produce patterned courtship songs to increase their chance of success with a female. For some animals, song structure can vary considerably from one rendition to the next; neural noise within pattern generating circuits is widely assumed to be the primary source of such variability, and statistical models that incorporate neural noise are successful at reproducing the full variation present in natural songs. In direct contrast, this study demonstrates that much of the pattern variability in Drosophila courtship song can be explained by taking into account the dynamic sensory experience of the male. In particular, using a quantitative behavioural assay combined with computational modelling, this stuyd found that males use fast modulations in visual and self-motion signals to pattern their songs, a relationship that was shown to be evolutionarily conserved. Using neural circuit manipulations, the pathways involved in song patterning choices were identified, and females were shown to be sensitive to song features. These data not only demonstrate that Drosophila song production is not a fixed action pattern, but establish Drosophila as a valuable new model for studies of rapid decision-making under both social and naturalistic conditions.

Gomez-Marin, A. and Louis, M. (2014). Multilevel control of run orientation in Drosophila larval chemotaxis. Front Behav Neurosci 8: 38. PubMed ID: 24592220
Summary: Chemotaxis is a powerful paradigm to study how orientation behavior is driven by sensory stimulation. Drosophila larvae navigate odor gradients by controlling the duration of their runs and the direction of their turns. Straight runs and wide-amplitude turns represent two extremes of a behavioral continuum. This study establishes that, on average, runs curl toward the direction of higher odor concentrations. The orientation and strength of the local odor gradient perpendicular to the direction of motion was found to modulate the orientation of individual runs in a gradual manner. How this error-correction mechanism, called weathervaning, contributes to larval chemotaxis is discussed. Larvae with a genetically modified olfactory system were used to demonstrate that unilateral function restricted to a single olfactory sensory neuron (OSN) is sufficient to direct weathervaning. The finding that bilateral sensing is not necessary to control weathervaning highlights the role of temporal sampling. A correlational analysis between sensory inputs and behavioral outputs suggests that weathervaning results from low-amplitude head casts implemented without interruption of the run. In addition, the involvement of a sensorimotor memory arising from previous reorientation events is reported. Together, these results indicate that larval chemotaxis combines concurrent orientation strategies that involve complex computations on different timescales.

Saturday, March 8th

Oliva, C., Choi, C. M., Nicolai, L. J., Mora, N., De Geest, N. and Hassan, B. A. (2014). Proper connectivity of Drosophila motion detector neurons requires Atonal function in progenitor cells. Neural Dev 9: 4. PubMed ID: 24571981
Summary: Vertebrates and invertebrates obtain visual motion information by channeling moving visual cues perceived by the retina through specific motion sensitive synaptic relays in the brain. In Drosophila, the series of synaptic relays forming the optic lobe are known as the lamina, medulla, lobula and lobula plate neuropiles. The fly's motion detection output neurons, called the T4 and T5 cells, reside in the lobula plate. Adult optic lobe neurons are derived from larval neural progenitors in two proliferating compartments known as the outer and inner proliferation centers (OPC and IPC). Important insight has been gained into molecular mechanisms involved in the development of the lamina and medulla from the OPC, though less is known about the development of the lobula and lobula plate. This study shows that the proneural gene Atonal is expressed in a subset of IPC progenitors that give rise to the higher order motion detection neurons, T4 and T5, of the lobula plate. Atonal does not act as a proneural gene in this context. Rather, it is required specifically in IPC neural progenitors to regulate neurite outgrowth in the neuronal progeny. These findings reveal that a proneural gene is expressed in progenitors but is required for neurite development of their progeny neurons. This suggests that transcriptional programs initiated specifically in progenitors are necessary for subsequent neuronal morphogenesis.

Bates, K. E., Sung, C., Hilson, L. and Robinow, S. (2014). unfulfilled Interacting Genes Display Branch-Specific Roles in the Development of Mushroom Body Axons in Drosophila melanogaster. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 24558265
Summary: The mushroom body (MB) of Drosophila is an organized collection of interneurons that is required for learning and memory. Each of the three subtypes of MB neurons, γ, α'/β', and α/β, branch at some point during their development, providing an excellent model in which to study the genetic regulation of axon branching. Given the sequential birth order and the unique patterning of MB neurons, it is likely that specific gene cascades are required for the different guidance events that form the characteristic lobes of the MB. The nuclear receptor Unfulfilled (Unf), a transcription factor, is required for the differentiation of all MB neurons. A classical genetic suppressor screen that takes advantage of the fact that ectopic expression of unf causes lethality was developed and utilized to identify candidate genes that act downstream of Unf. It was hypothesized that reducing the copy number of unf-interacting genes will suppress the unf-induced lethality. Nineteen candidate genes were identified that when mutated suppress the unf-induced lethality. To test whether candidate genes impact MB development, a secondary phenotypic screen was performed in which the morphologies of the MBs in animals heterozygous for unf and a specific candidate gene were analyzed. Medial MB lobes were thin, missing, or misguided dorsally in five double heterozygote combinations (unf/+;axin/+, unf/+;Fps85D/+, unf/+;Tsc1/+, unf/+;Rheb/+, ;unf/+;msn/+). Dorsal MB lobes were missing in ;unf/+;DopR2/+ or misprojecting beyond the termination point in unf/+;Sytβ double heterozygotes. These data suggest that unf and unf-interacting genes play specific roles in axon development in a branch-specific manner.

Peled, E. S., Newman, Z. L. and Isacoff, E. Y. (2014). Evoked and Spontaneous Transmission Favored by Distinct Sets of Synapses. Curr Biol 24(5): 484-93. PubMed ID: 24560571
Summary: Spontaneous 'miniature' transmitter release takes place at low rates at all synapses. Long thought of as an unavoidable leak, spontaneous release has recently been suggested to be mediated by distinct pre- and postsynaptic molecular machineries and to have a specialized role in setting up and adjusting neuronal circuits. It remains unclear how spontaneous and evoked transmission are related at individual synapses, how they are distributed spatially when an axon makes multiple contacts with a target, and whether they are commonly regulated. Electrophysiological recordings in the Drosophila larval neuromuscular junction, in the presence of the use-dependent glutamate receptor (GluR) blocker philanthotoxin, indicated that spontaneous and evoked transmission employ distinct sets of GluRs. In vivo imaging of transmission using synaptically targeted GCaMP3 to detect Ca2+ influx through the GluRs revealed little spatial overlap between synapses participating in spontaneous and evoked transmission. Spontaneous and evoked transmission were oppositely correlated with presynaptic levels of the protein Brp: synapses with high Brp favored evoked transmission, whereas synapses with low Brp were more active spontaneously. High-frequency stimulation did not increase the overlap between evoked and spontaneous transmission, and instead decreased the rate of spontaneous release from synapses that were highly active in evoked transmission. It is concluded that although individual synapses can participate in both evoked and spontaneous transmission, highly active synapses show a preference for one mode of transmission. The presynaptic protein Brp promotes evoked transmission and suppresses spontaneous release. These findings suggest the existence of presynaptic mechanisms that promote synaptic specialization to either evoked or spontaneous transmission.

Bemben, M. A., Shipman, S. L., Hirai, T., Herring, B. E., Li, Y., Badger, J. D., Nicoll, R. A., Diamond, J. S. and Roche, K. W. (2014). CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses. Nat Neurosci 17: 56-64. PubMed ID: 24336150
Summary: Neuroligins (see Drosophila Neuroligins) are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII; see Drosophila CaMKII). This study reports that CaMKII robustly phosphorylates the intracellular domain of NL-1. T739 was shown to be the dominant CaMKII site on NL-1, and it is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. These results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.

Friday, March 7th

Roy, S., Huang, H., Liu, S. and Kornberg, T. B. (2014). Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein. Science 343: 1244624. PubMed ID: 24385607
Summary: Decapentaplegic (Dpp), a Drosophila morphogen signaling protein, transfers directly at synapses made at sites of contact between cells that produce Dpp and cytonemes that extend from recipient cells. The Dpp that cytonemes receive moves together with activated receptors toward the recipient cell body in motile puncta. Genetic loss-of-function conditions for diaphanous, shibire, neuroglian, and capricious perturbed cytonemes by reducing their number or only the synapses they make with cells they target, and reduced cytoneme-mediated transport of Dpp and Dpp signaling. These experiments provide direct evidence that cells use cytonemes to exchange signaling proteins, that cytoneme-based exchange is essential for signaling and normal development, and that morphogen distribution and signaling can be contact-dependent, requiring cytoneme synapses.

de la Cova, C., Senoo-Matsuda, N., Ziosi, M., Wu, D. C., Bellosta, P., Quinzii, C. M. and Johnston, L. A. (2014). Supercompetitor Status of Drosophila Myc Cells Requires p53 as a Fitness Sensor to Reprogram Metabolism and Promote Viability. Cell Metab [Epub ahead of print]. PubMed ID: 24561262
Summary: In growing tissues, cell fitness disparities can provoke interactions that promote stronger cells at the expense of the weaker in a process called cell competition. The mechanistic definition of cell fitness is not understood, nor is it understood how fitness differences are recognized. Drosophila cells with extra Myc activity acquire 'supercompetitor' status upon confrontation with wild-type (WT) cells, prompting the latter's elimination via apoptosis. This study shows that such confrontation enhances glycolytic flux in Myc cells and promotes their fitness and proliferation in a p53-dependent manner. Whereas p53 loss in noncompeting Myc cells is inconsequential, its loss impairs metabolism, reduces viability, and prevents the killing activity of Myc supercompetitor cells. It is proposed that p53 acts as a general sensor of competitive confrontation to enhance the fitness of the 'winner' population. These findings suggest that the initial confrontation between precancerous and WT cells could enhance cancer cell fitness and promote tumor progression.

Escalante, A., Murillo, B., Morenilla-Palao, C., Klar, A. and Herrera, E. (2013). Zic2-dependent axon midline avoidance controls the formation of major ipsilateral tracts in the CNS. Neuron 80: 1392-1406. PubMed ID: 24360543
Summary: In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. This study demonstrates that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2 (see Drosophila ). In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 (see Drosophila Ephrin) expression in dorsospinal neurons to prevent midline crossing while Robo3 (see Drosophila Roundabout) is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, these data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways.

Ferreira, T., Wilson, S. R., Choi, Y. G., Risso, D., Dudoit, S., Speed, T. P. and Ngai, J. (2014). Silencing of Odorant Receptor Genes by G Protein betagamma Signaling Ensures the Expression of One Odorant Receptor per Olfactory Sensory Neuron. Neuron 81: 847-859. PubMed ID: 24559675
Summary: Olfactory sensory neurons express just one out of a possible approximately 1,000 odorant receptor genes, reflecting an exquisite mode of gene regulation. In one model, once an odorant receptor is chosen for expression, other receptor genes are suppressed by a negative feedback mechanism, ensuring a stable functional identity of the sensory neuron for the lifetime of the cell. The signal transduction mechanism subserving odorant receptor gene silencing remains obscure, however. This study demonstrates in the zebrafish that odorant receptor gene silencing is dependent on receptor activity. Moreover, signaling through G protein βγ subunits (see Drosophila Heterotrimeric G-proteins) is both necessary and sufficient to suppress the expression of odorant receptor genes and likely acts through histone methylation to maintain the silenced odorant receptor genes in transcriptionally inactive heterochromatin. These results link receptor activity with the epigenetic mechanisms responsible for ensuring the expression of one odorant receptor per olfactory sensory neuron.

Thursday, March 6th

Lacin, H., Rusch, J., Yeh, R. T., Fujioka, M., Wilson, B. A., Zhu, Y., Robie, A. A., Mistry, H., Wang, T., Jaynes, J. B. and Skeath, J. B. (2014). Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of Nitric Oxide Synthase and Fd59a/Fox-D. Dev Biol [Epub ahead of print]. PubMed ID: 24512689
Summary: Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, and Tail-up (Islet) to guide the stereotyped differentiation, connectivity, and function of a subset of neurons in Drosophila. The role of Hb9 in directing neuronal differentiation is well documented, but the lineage of Hb9+ neurons is only partly characterized, its regulation is poorly understood, and most of the downstream genes through which it acts remain at large. This study completes the lineage tracing of all embryonic Hb9+ neurons (to eight neuronal lineages) and provides evidence that hb9, lim3, and tail-up are coordinately regulated by a common set of upstream factors. Through the parallel use of micro-array gene expression profiling and the Dam-ID method, Hb9-regulated genes were sought, uncovering transcription factors as the most over-represented class of genes regulated by Hb9 (and Nkx6) in the CNS. By a nearly ten-to-one ratio, Hb9 represses rather than activates transcription factors, highlighting transcriptional repression of other transcription factors as a core mechanism by which Hb9 governs neuronal determination. From the small set of genes activated by Hb9, the expression and function of two were characterized - fd59a/foxd, which encodes a transcription factor, and Nitric oxide synthase. Under standard lab conditions, both genes are dispensable for Drosophila development, but Nos appears to inhibit hyper-active behavior and fd59a appears to act in octopaminergic neurons to control egg-laying behavior. Together these data clarify the mechanisms through which Hb9 governs neuronal specification and differentiation and provide an initial characterization of the expression and function of Nos and fd59a in the Drosophila CNS.

Zarin, A. A., Asadzadeh, J., Hokamp, K., McCartney, D., Yang, L., Bashaw, G. J. and Labrador, J. P. (2014). A transcription factor network coordinates attraction, repulsion, and adhesion combinatorially to control motor axon pathway selection. Neuron [Epub ahead of print]. PubMed ID: 24560702
Summary: Combinations of transcription factors (TFs) instruct precise wiring patterns in the developing nervous system; however, how these factors impinge on surface molecules that control guidance decisions is poorly understood. Using mRNA profiling, the complement of membrane molecules regulated by the homeobox TF Even-skipped (Eve), the major determinant of dorsal motor neuron (dMN) identity in Drosophila, was identifed. Combinatorial loss- and gain-of-function genetic analyses of Eve target genes indicate that the integrated actions of attractive, repulsive, and adhesive molecules direct eve-dependent dMN axon guidance. Furthermore, combined misexpression of Eve target genes is sufficient to partially restore CNS exit and can convert the guidance behavior of interneurons to that of dMNs. Finally, it was shown that a network of TFs, comprised of eve, zfh1, and grain, induces the expression of the Unc5 and Beaten-path guidance receptors and the Fasciclin 2 and Neuroglian adhesion molecules to guide individual dMN axons (Zarin, 2013).

Andres, M., Turiegano, E., Gopfert, M. C., Canal, I. and Torroja, L. (2014). The Extracellular Matrix Protein Artichoke Is Required for Integrity of Ciliated Mechanosensory and Chemosensory Organs in Drosophila Embryos. Genetics [Epub ahead of print]. PubMed ID: 24496014
Summary: Sensory cilia are often encapsulated by an extracellular matrix (ECM). In Caenorhabditis elegans, Drosophila melanogaster, and vertebrates, this ECM is thought to be directly involved in ciliary mechanosensing by coupling external forces to the ciliary membrane. Drosophila mechano- and chemosensory cilia are both associated with an ECM, indicating that the ECM may have additional roles that go beyond mechanosensory cilium function. This study identified Artichoke (ATK: CG5195), an evolutionary conserved leucine-rich repeat ECM protein that is required for normal morphogenesis and function of ciliated sensilla in Drosophila. atk is transiently expressed in accessory cells in all ciliated sensory organs during their late embryonic development. Antibody stainings show ATK protein in the ECM that surrounds sensory cilia. Loss of ATK protein in atk null mutants leads to cilium deformation and disorientation in chordotonal organs, apparently without uncoupling the cilia from the ECM, and consequently to locomotion defects. Moreover, impaired chemotaxis in atk mutant larvae suggests that, based on ATK protein localization, the ECM is also crucial for the correct assembly of chemosensory receptors. In addition to defining a novel ECM component, these findings show the importance of ECM integrity for the proper morphogenesis of ciliated organs in different sensory modalities.

Biffar, L. and Stollewerk, A. (2014). Conservation and evolutionary modifications of neuroblast expression patterns in insects. Dev Biol [Epub ahead of print]. PubMed ID: 24525296
Summary: One of the major questions in evolutionary developmental neurobiology is how neuronal networks have been adapted to different morphologies and behaviour during evolution. Analyses of neurogenesis in representatives of all arthropod species have revealed evolutionary modifications of various developmental mechanisms. Among others, variations can be seen in mechanisms that are associated with changes in neural progenitor identity, which in turn determines the neuronal subtype of their progeny. Comparative analyses of the molecular processes that underlie the generation of neuronal identity might therefore uncover the steps of evolutionary changes that eventually resulted in modifications in neuronal networks. This study addressed this question in the flour beetle Tribolium castaneum by analyzing and comparing the development and expression profile of neural stem cells (neuroblasts) to the published neuroblast map of the fruit fly Drosophila melanogaster. Substantial changes in the identity of neuroblasts have occurred during insect evolution. In almost all neuroblasts the relative positions in the ventral hemi-neuromeres are conserved; however, in over half of the neuroblasts the time of formation as well as the gene expression profile has changed. The neuroblast map presented in this study can be used for future comparative studies on individual neuroblast lineages in D. melanogaster and T. castaneum and additional markers and information on lineages can be added. These data suggest that evolutionary changes in the expression profile of individual neuroblasts might have contributed to the evolution of neural diversity and subsequently to changes in neuronal networks in arthropod.

Wednesday, March 5th

Papagiannouli, F., Schardt, L., Grajcarek, J., Ha, N. and Lohmann, I. (2014). The Hox gene abd-B controls stem cell niche function in the Drosophila testis. Dev Cell 28: 189-202. PubMed ID: 24480643
Summary: Proper niche architecture is critical for stem cell function, yet only few upstream regulators are known. This study reports that the Hox transcription factor Abdominal-B (Abd-B), active in premeiotic spermatocytes of Drosophila testes, is essential for positioning the niche to the testis anterior by regulating integrin in neighboring somatic cyst cells. Abd-B also non-cell-autonomously controls critical features within the niche, including centrosome orientation and division rates of germline stem cells. By using genome-wide binding studies, it was found that Abd-B mediates its effects on integrin localization by directly controlling at multiple levels the signaling activity of the Sev ligand Boss via its direct targets src42A and sec63, two genes involved in protein trafficking and recycling. The data show that Abd-B, through local signaling between adjacent cell types, provides positional cues for integrin localization, which is critical for placement of the distant stem cell niche and stem cell activity.

Clough, E., Tedeschi, T. and Hazelrigg, T. (2014). Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1. Dev Biol [Epub ahead of print]. PubMed ID: 24485852
Summary: The Drosophila melanogaster histone lysine methyltransferase (HKMT) Eggless (Egg/dSETDB1) catalyzes methylation of Histone H3 lysine 9 (H3K9), a signature of repressive heterochromatin. Previous studies showed that H3K9 methylation by Egg is required for oogenesis. This study analyzed a set of EMS-induced mutations in the egg gene, identified the molecular lesions of these mutations, and compared the effects on oogenesis of both strong loss-of-function and weak hypomorphic alleles. These studies show that H3K9 methylation by Egg is required for multiple stages of oogenesis. Mosaic expression experiments show that the egg gene is not required intrinsically in the germ cells for their early differentiation, but is required in the germ cells for their survival past stage 5 of oogenesis. egg is also required in germ stem cells for their maintenance, since egg- germ stem cells initially survive but are not maintained as females age. Mosaic analysis also reveals that the early egg chamber budding defects in egg- ovaries are due to an intrinsic requirement for egg in follicle stem cells and their descendents, and that egg plays a non-autonomous role in somatic cells in the germarium to influence the differentiation of early germ cells.

Yan, D., Neumuller, R. A., Buckner, M., Ayers, K., Li, H., Hu, Y., Yang-Zhou, D., Pan, L., Wang, X., Kelley, C., Vinayagam, A., Binari, R., Randklev, S., Perkins, L. A., Xie, T., Cooley, L. and Perrimon, N. (2014). A regulatory network of Drosophila germline stem cell self-renewal. Dev Cell 28: 459-473. PubMed ID: 24576427
Summary: Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, a large-scale RNAi screen was performed in Drosophila female germline stem cells (GSCs) covering approximately 25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, the histone methyltransferase Set1 was identified as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, this study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.

Green, D. A. and Extavour, C. G. (2014). Insulin signalling underlies both plasticity and divergence of a reproductive trait in Drosophila. Proc Biol Sci 281: 20132673. PubMed ID: 24500165
Summary: Phenotypic plasticity is the ability of a single genotype to yield distinct phenotypes in different environments. The molecular mechanisms linking phenotypic plasticity to the evolution of heritable diversification, however, are largely unknown. This study shows that insulin/insulin-like growth factor signalling (IIS) underlies both phenotypic plasticity and evolutionary diversification of ovariole number, a quantitative reproductive trait, in Drosophila. IIS activity levels and sensitivity have diverged between species, leading to both species-specific ovariole number and species-specific nutritional plasticity in ovariole number. Plastic range of ovariole number correlates with ecological niche, suggesting that the degree of nutritional plasticity may be an adaptive trait. This demonstrates that a plastic response conserved across animals can underlie the evolution of morphological diversity, underscoring the potential pervasiveness of plasticity as an evolutionary mechanism.

Tuesday March 4th

Lin, A. C., Bygrave, A. M., de Calignon, A., Lee, T. and Miesenbock, G. (2014). Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination. . Nat Neurosci [Epub ahead of print]. PubMed ID: 24561998
Summary: Sparse coding may be a general strategy of neural systems for augmenting memory capacity. In Drosophila melanogaster, sparse odor coding by the Kenyon cells of the mushroom body is thought to generate a large number of precisely addressable locations for the storage of odor-specific memories. However, it remains untested how sparse coding relates to behavioral performance. This study demonstrates that sparseness is controlled by a negative feedback circuit between Kenyon cells and the GABAergic anterior paired lateral (APL) neuron. Systematic activation and blockade of each leg of this feedback circuit showed that Kenyon cells activated APL and APL inhibited Kenyon cells. Disrupting the Kenyon cell-APL feedback loop decreased the sparseness of Kenyon cell odor responses, increased inter-odor correlations and prevented flies from learning to discriminate similar, but not dissimilar, odors. These results suggest that feedback inhibition suppresses Kenyon cell activity to maintain sparse, decorrelated odor coding and thus the odor specificity of memories.

Shen, K., Tootoonian, S. and Laurent, G. (2013). Encoding of mixtures in a simple olfactory system. Neuron 80: 1246-1262. PubMed ID: 24210905
Summary: Natural odors are usually mixtures; yet, humans and animals can experience them as unitary percepts. Olfaction also enables stimulus categorization and generalization. How these computations are performed were studied with the responses of 168 locust antennal lobe projection neurons (PNs) to varying mixtures of two monomolecular odors, and of 174 PNs and 209 mushroom body Kenyon cells (KCs) to mixtures of up to eight monomolecular odors. Single-PN responses showed strong hypoadditivity and population trajectories clustered by odor concentration and mixture similarity. KC responses were much sparser on average than those of PNs and often signaled the presence of single components in mixtures. Linear classifiers could read out the responses of both populations in single time bins to perform odor identification, categorization, and generalization. These results suggest that odor representations in the mushroom body may result from competing optimization constraints to facilitate memorization (sparseness) while enabling identification, classification, and generalization.

Kato, S., Xu, Y., Cho, C. E., Abbott, L. F. and Bargmann, C. I. (2014). Temporal Responses of C. elegans Chemosensory Neurons Are Preserved in Behavioral Dynamics. Neuron 81: 616-628. PubMed ID: 24440227
Summary: Animals track fluctuating stimuli over multiple timescales during natural olfactory behaviors. This study defines mechanisms underlying these computations in Caenorhabditis elegans. By characterizing neuronal calcium responses to rapidly fluctuating odor sequences, it was shown that sensory neurons reliably track stimulus fluctuations relevant to behavior. AWC olfactory neurons respond to multiple odors with subsecond precision required for chemotaxis, whereas ASH nociceptive neurons integrate noxious cues over several seconds to reach a threshold for avoidance behavior. Each neuron's response to fluctuating stimuli is largely linear and can be described by a biphasic temporal filter and dynamical model. A calcium channel mutation alters temporal filtering and avoidance behaviors initiated by ASH on similar timescales. A sensory G-alpha protein mutation affects temporal filtering in AWC and alters steering behavior in a way that supports an active sensing model for chemotaxis. Thus, temporal features of sensory neurons can be propagated across circuits to specify behavioral dynamics.

Donlea, J. M., Pimentel, D. and Miesenbock, G. (2014). Neuronal machinery of sleep homeostasis in Drosophila. Neuron 81: 860-872. PubMed ID: 24559676
Summary: Sleep is under homeostatic control, but the mechanisms that sense sleep need and correct sleep deficits remain unknown. This study reports that sleep-promoting neurons with projections to the dorsal fan-shaped body (FB) form the output arm of Drosophila's sleep homeostat. Homeostatic sleep control requires the Rho-GTPase-activating protein encoded by the crossveinless-c (cv-c) gene in order to transduce sleep pressure into increased electrical excitability of dorsal FB neurons. cv-c mutants exhibit decreased sleep time, diminished sleep rebound, and memory deficits comparable to those after sleep loss. Targeted ablation and rescue of Cv-c in sleep-control neurons of the dorsal FB impair and restore, respectively, normal sleep patterns. Sleep deprivation increases the excitability of dorsal FB neurons, but this homeostatic adjustment is disrupted in short-sleeping cv-c mutants. Sleep pressure thus shifts the input-output function of sleep-promoting neurons toward heightened activity by modulating ion channel function in a mechanism dependent on Cv-c.

Monday, March 3rd

Fan, Y., Wang, S., Hernandez, J., Yenigun, V. B., Hertlein, G., Fogarty, C. E., Lindblad, J. L. and Bergmann, A. (2014). Genetic Models of Apoptosis-Induced Proliferation Decipher Activation of JNK and Identify a Requirement of EGFR Signaling for Tissue Regenerative Responses in Drosophila. PLoS Genet 10: e1004131. PubMed ID: 24497843
Summary: Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, an efficient assay was developed for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. A subset of genes was identified acting upstream of JNK activity including Rho1. It was also demonstrated that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, signaling by the EGFR pathway was identified as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation.

Kim, H. J., Raphael, A. R., Ladow, E. S., McGurk, L., Weber, R. A., Trojanowski, J. Q., Lee, V. M., Finkbeiner, S., Gitler, A. D. and Bonini, N. M. (2014). Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models. Nat Genet 46: 152-160. PubMed ID: 24336168
Summary: Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 (Drosophila homolog TAR DNA-binding protein-43 homolog) and ataxin-2 (see Drosophila Ataxin-2), is that they localize to stress granules. Unexpectedly, this study found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2alpha phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. The efficacy of a small molecule inhibitor of eIF2alpha phosphorylation was investigated in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

Machamer, J. B., Collins, S. E. and Lloyd, T. E. (2014). The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24569165
Summary: Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5-10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. This study investigated the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compare them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). Lethality and locomotor phenotypes were found to correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons.

Frost, B., Hemberg, M., Lewis, J. and Feany, M. B. (2014). Tau promotes neurodegeneration through global chromatin relaxation. Nat Neurosci 17(3): 357-66. PubMed ID: 24464041
Summary: The microtubule-associated protein tau is involved in a number of neurodegenerative disorders, including Alzheimer's disease. Previous studies have linked oxidative stress and subsequent DNA damage to neuronal death in Alzheimer's disease and related tauopathies. Given that DNA damage can substantially alter chromatin structure, this study examined epigenetic changes in tau-induced neurodegeneration. Widespread loss was found of heterochromatin in tau transgenic Drosophila and mice and in human Alzheimer's disease. Notably, genetic rescue of tau-induced heterochromatin loss substantially reduced neurodegeneration in Drosophila. Oxidative stress and subsequent DNA damage were identified as a mechanistic link between transgenic tau expression and heterochromatin relaxation, and heterochromatin loss was found to permit aberrant gene expression in tauopathies. Furthermore, large-scale analyses from the brains of individuals with Alzheimer's disease revealed a widespread transcriptional increase in genes that were heterochromatically silenced in controls. These results establish heterochromatin loss as a toxic effector of tau-induced neurodegeneration and identify chromatin structure as a potential therapeutic target in Alzheimer's disease.

Sunday, March 2nd

Tufi, R., et al. (2014). Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson's disease. Nat Cell Biol 16(2): 157-66. PubMed ID: 24441527
Summary: Mutations in PINK1 cause early-onset Parkinson's disease (PD). Studies in Drosophila have highlighted mitochondrial dysfunction on loss of Pink1 as a central mechanism of PD pathogenesis. This study shows that global analysis of transcriptional changes in Drosophila pink1 mutants reveals an upregulation of genes involved in nucleotide metabolism, critical for neuronal mitochondrial DNA synthesis. These key transcriptional changes were also detected in brains of PD patients harbouring PINK1 mutations. Genetic enhancement of the nucleotide salvage pathway in neurons of pink1 mutant flies rescues mitochondrial impairment. In addition, pharmacological approaches enhancing nucleotide pools reduce mitochondrial dysfunction caused by Pink1 deficiency. It is concluded that loss of Pink1 evokes the activation of a previously unidentified metabolic reprogramming pathway to increase nucleotide pools and promote mitochondrial biogenesis. It is proposed that targeting strategies enhancing nucleotide synthesis pathways may reverse mitochondrial dysfunction and rescue neurodegeneration in PD and, potentially, other diseases linked to mitochondrial impairment.

Klein, P., Muller-Rischart, A. K., Motori, E., Schonbauer, C., Schnorrer, F., Winklhofer, K. F. and Klein, R. (2014). Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J 33(4): 341-55. PubMed ID: 24473149
Summary: Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. This study investigated whether the Drosophila homolog of Ret, Ret oncogene, can rescue Pink1 and park mutant phenotypes. A signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.

Debattisti, V., Pendin, D., Ziviani, E., Daga, A. and Scorrano, L. (2014). Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion. J Cell Biol 204(3): 303-12. PubMed ID: 24469638
Summary: Ablation of the mitochondrial fusion and endoplasmic reticulum (ER)-tethering protein Mfn2 causes ER stress, but whether this is just an epiphenomenon of mitochondrial dysfunction or a contributor to the phenotypes in Mitofusin (Mfn)-depleted Drosophila melanogaster is unclear. This paper shows that reduction of ER dysfunction ameliorates the functional and developmental defects of flies lacking the single Mfn mitochondrial assembly regulatory factor (Marf). Ubiquitous or neuron- and muscle-specific Marf ablation was lethal, altering mitochondrial and ER morphology and triggering ER stress that was conversely absent in flies lacking the fusion protein Optic atrophy 1. Expression of Mfn2 and ER stress reduction in flies lacking Marf corrected ER shape, attenuating the developmental and motor defects. Thus, ER stress is a targetable pathogenetic component of the phenotypes caused by Drosophila Mfn ablation.

Azuma, Y., et al. (2014). Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24497576
Summary: In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) (CG3606) is the Drosophila ortholog of human FUS. Previous studies have established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons at NMJs; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause motoneuron degeneration similar to that seen in FUS-related ALS. This study aimed to explore molecules that affect these ALS-like phenotypes of Drosophila models with eye-specific and neuron-specific Caz-knockdown. Several previously reported ALS-related genes were examined and genetic links were found between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function-allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the motoneuron-degenetation phenotype caused by Caz-knockdown. Conversely, overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. These data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. The results indicate that therapeutic agents that upregulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of motoneurons in ALS.

Saturday, March 1st

Bulow, M. H., Bulow, T. R., Hoch, M., Pankratz, M. J. and Junger, M. A. (2014). Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity. Sci Rep 4: 4048. PubMed ID: 24513978
Summary: Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, this study investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, evidence is provided that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells.

Ignesti, M., Barraco, M., Nallamothu, G., Woolworth, J. A., Duchi, S., Gargiulo, G., Cavaliere, V. and Hsu, T. (2014). Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23. BMC Biol 12: 12. PubMed ID: 24528630
Summary: The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. This study analyzed the role of Awd in controlling Notch signaling during development. To study the awd gene function, genetic mosaic approaches were used to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. The results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments it was shown that Notch receptor accumulates in early endosome in awd mutant follicle cells. Trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation. This report has demonstrated that awd is essential for Notch signaling via its endocytic role. In addition this study has identified the endocytic step at which Awd function is required for Notch signaling and evidence was obtained indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.

Simoes Sde, M., Mainieri, A. and Zallen, J. A. (2014). Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension. J Cell Biol 204: 575-589. PubMed ID: 24535826
Summary: Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. This study shows that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.

Laflamme, B. A., Avila, F. W., Michalski, K. and Wolfner, M. F. (2014). A Drosophila protease cascade member, Seminal Metalloprotease-1, is activated stepwise by male factors and requires female factors for full activity. Genetics [Epub ahead of print]. PubMed ID: 24514904
Summary: Females and males of sexually reproducing animals must cooperate at the molecular and cellular level for fertilization to succeed, even though some aspects of reproductive molecular biology appear to involve antagonistic interactions. Previously studies have reported the existence of a proteolytic cascade in Drosophila melanogaster seminal fluid that is initiated in the male and ends in the female. This proteolytic cascade, which processes at least two seminal fluid proteins (Sfps), is a useful model for understanding the regulation of Sfp activities, including proteolysis cascades in mammals. This study investigated the activation mechanism of the downstream protease in the cascade, the astacin-family metalloprotease Seminal metalloprotease-1 (Semp1, CG11864), focusing on the relative contribution of the male and female to its activation. A naturally-occurring semp1 null mutation was identified within the Drosophila Genetic Reference Panel. By expressing mutant forms of Semp1 in males homozygous for the null mutation, it was discovered that cleavage is required for the complete activation of Semp1, and at least two sites were defined that are essential for this activational cleavage. These amino acid residues suggest a two-step mechanism for Semp1 activation, involving the action of at least two male-derived proteases. Although the cascade's substrates potentially influence both fertility and sperm competition within the mated female, the role of female factors in the activation or activity of Semp1 is unknown. This study shows that Semp1 can undergo its activational cleavage in male ejaculates, without female contributions, but that cleavage of Semp1's substrates does not proceed to completion in ejaculates, indicating an essential role for female factors in Semp1's full activity. In addition, it was found that expression of Semp1 in virgin females demonstrates that females can activate this protease on their own resulting in activity that is complete but substantially delayed.

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