Windows 2012 Remote Desktop Reconfig on MORGAN


MORGAN was running still the trial period remote desktop, which took some effort to get set up on Windows 2012 in the first place and was behaving a bit differently than on CAJAL. On CAJAL I had entered the license info on the original configuration run but it promptly forgot this info when the trial period expired (it thought it was still running on trial licenses) and I had to renter this info to get it to run. So on Morgan I just let the trial expire first before trying to install the licenses I bought.

Installing licenses

It’s really hard to find out where to enter the RD licenses. You might think this big licenses icon in the server manager would be the place…


but you’d be wrong. This doesn’t even have the option to enter yet.

So I first readded server roles, remote desktop (I think this was already working, see previous configure of remote desktop Morgan post). I don’t think this was necessary, but it went through a whole round of install and reboot. (during which time the monitor was disconnected and refused to reconnect).

What I really want is here: Select the RD icon at the bottom left of the tool panel in Server Manager, and then select Collections. We first need to create a new Session. I created a MorganSession here.


Then select Servers and right click on the Morgan Server and select RD License Manager. Here you will be able to enter the Agreement number and specify the number (5) and type (per User CAL). I actually did this before adding the Morgan Session and it didn’t work (even after a system reboot). Then I added the session and then in started working.

You can also test if the licenses are working here:



  • now the server works again and accepts remote connections.
  • only another hour+ of troubleshooting just to enter two stupid numbers in the right place.
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Tuesday 3/10/15

10:40 am – 8:30 pm

The little things

  • uploaded Fly Meeting Notes to notebook.
  • Score approximate fly ages in Georgia’s samples (see email)
  • reply to some outstanding emails from fly meeting
  • contact BWF about K99

Chromatin paper

  • reorganizing figures
  • working on Fig 2
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Monday, 3/9/15

9:30 am – 12:00 am


  • lab meeting (10 am – 1:20 pm)
  • discussion with GS about project data
  • discussion with Bogdan about paper organization

Cell culture

  • froze down 12 new vials of Kc stocks (in -80C at 11:00 pm).
  • skipped PBS rinse. Probably over-did density (50 mL instead of 30 mL).
  • passaged cells

Fly culture

  • new stocks arrived today

Data organization

  • starting new way of organizing data
  • saving panels of images
  • saving flists with other data indexed by excel sheet
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Fly Meeting, Sunday 3/8/15

Chris Rushlow


  • by 3 hrs, most of the cells know their fate (end of cc14).
  • MZT degradation of 1000s of maternal RNAs, activation of zygotic genome
  • zelda is in nuclei early, but levels rise abruptly after 1 hr post fertilization
  • regulates cellularization, sex determination, and patterning
  • dosal gradient uneffected but genes like sog substantially depleted.

helping Dl bind

  • ChIP-seq 2-3 hr wt and zld null embryos using anti-Dorsal. Dorsal ChIP peak at sog drops dramatically
  • most of 3500 Dl are bound by Zld
  • 20% decrease by 18% increase (Dl and Zld was already low
  • Question: take 2% strongest peaks, what happens?
  • heat maps: near TSS, no enriched Zld sites near these sites.
  • decrease group is more frequent at intron and intergeneic regions than at promoter. STAT9E, Dl, Cad, Bcd, Twi sites. (Bcd binding also shown to reduce)

How does Zld help these factors bind?

  • Pioneer TF factor?
    • Li et al 2014, Yanez-Cuna 2012, Xu et al 2014
    • binds early, bind before histone mods.
  • MNase seq
  • are zelda bound regions enriched for early enhancers? (e.g. REDfly enhancers)
  • Defined 1031 ‘early’ enhancers.
  • early enriched enhancer, Dl bound regions, and HOT regions all enriched in the top 1000 (of 6000) Zld peaks.
  • wt MNase show clear deplition at broad Zld summits, which is lost in zld null (also shown with FAIRE and anti-H3 by Harrison and Eisen labs)
  • Nulceosome model predicition from sequence suggests that Zld binds positioned nucleosomes
  • Sort 6000 regions by most open — these are not that much more zld bound.
    • slightly enriched in HOT regions.
    • depleted in early enhancers
    • enriched in strongly positioned nuclesomes lacking flanks’
  • low nucleosome occcupancy per se does not correlate with enhancers.

Can Zelda bind its motifs in nucleosomes?

  • constitute mono-nucleosomes from S2 cells
  • add zelda, see a higher order shift (zelda binds nucleosome rich DNA)’
  • can anti-H3 show it’s there in both bands
  • Is it displacing nucleosomes? Or why do you see H3 in the shifted mononucleosome band?

Explaining enhancer activity

  • many of these genes can still kick on, but are delayed.
  • Human homologs (by functin?) Pou5f1 SoxB1 Nanong in Fish pioneer factors. Homologs of Yamanka cellular reprograming factors.
  • Zelda doesn’t activate itself

Yukiko Yamashita: asymmetric stem cell division in the testes

  • how is assymetric stem cell division regulated?
  • hypothesis – niche provides limited range signalling, so that only proximal cells remain stem. Does niche orient division plane?
  • Upd puncta mark the hub cell (specifying the niche)
  • observe ~1.2 puncta per cell branching off the hub. punta tend to align with spindle poles
  • laser oblate this puncta, centrioles / spindle fiber of stem cells are no longer oriented perpindcular to the cell reliably.
  • knock down Upd in the hub cells, see increased spindle misorientation.

Mechanisms and Functions of RNA Silencing Pathways Phillip Zamore, RNA Therapeutics Institute, University of Massachusetts Medical School

  • how cells silence transposons in the germline with pi RNA
  • antisence primary piRNAs bind Piwi and go on to silence transposns in chromatin
  • Ping-pong model: mature piRNA is a secondary piRNA (5′ end generated by Piwi). amplifies original antisenste piRNA. depends on aubergene.
  • most piRNAs are synthesized at the end of another piRNA (most frequent gap is 0) This sequential production is Zucchini dependent and is particular to Piwi bound piRNAs (not aubergne bound ones)
  • degradome fragments that map to transposons very preferentially (57%) begin with U (as do piRNAs)
  • aubergene is much more important to get silencing than piwi.
  • propose transcriptional silencing is prevelant (rather than previous model of mRNA degradation). RNA degradation is just a templating thing to build more silencer.
  • akin to spreading of small RNAs in plants and worms – spreading of piRNAs that are phased by another piRNA allows novel piRNA (something like that).

Decapentaplegic and the Control of Growth in the Drosophila Wing Imaginal Disc Matthew Gibson, Stowers Institute for Medical Research,

  • Morgan speculated about morphogen gradients from observations on vertebrate development
  • Turing patterns: Turing coins the term morphogen as substances that can confer form.
  • Wolpert put this into a cellular context in 1968: Towards a theoretical biology (french flag model)
  • ~90 year history before we got a handle on what they were, Fly plaid a central role
  • dpp acts at long range in the wing disk.
  • dpp gradient also controls wing growth. The mechanism is (hotly) debated
  • Growth in the disk is uniform.
  • Existing models:
    1. gradient sensor – uniform gradient uniform growth
    2. temporal change sensor – as levels increase steadily, steady growth
    3. brk repression leads to uniform growth
  • dpp[d12] is a regulatory mutant that doesn’t effect early embryo.
  • wing disks carrying dpp[d12] have no growth phenotype
  • detect loss of dpp in wing blade.

Flies and Alcohol: Interplay of Nature and Nurture Ulrike Heberlein

  • 1 in 12 adults have alcohol abuse or addiction
  • costs billions of dollars in economic damage
  • yeast sugar and hence ethanol have been around a long time
  • Robert Dudley has hypothesized our ancestors have evolved in presence of alcohol for a long time (drunken monkey hypothesis)
  • drunk fly behavior – first become hyperactive, then sedate, uncoordinated, and then pass out.
  • conserved response.
  • ID candidate genes for how ethanol functions in the nervous system.
  • look for mutants that inibreate sooner. ‘cheapdate’ gene chpd (student Monica Moore named the gene after herself)
  • flies develop gradually increasing preference for ethanol.
  • flies will develop the preference for alcohol even if laced with quinine (very bitter, flies don’t like)
  • relapse behavior — quickly reaquire high levels of consumption after being forced to abstain.
  • flies develop a preference for odor that has been paired with ethanol. remember this for a week. flies perfer the odor that predicts existence of alcohol even if they learn they have to go through electric shock to get there.
  • Flies develop tolerance over time
  • Flies show withdrawl symptoms if with-held from alcohol
  • human alcoholism — ~50% genetic risk ~50% environment
  • 80% of genes ID’d in flies also affect alcoholism in rats
  • What about Nuture / Envi experience?
  • sexually rejected male flies drink more
  • rejected males stop courting (mated females) and stop courting even other virgins
  • mated males start with an aversion and more to a weak preference
  • rejected males start with high preference
  • Mechanism?
  • rodants fles and humans link NPF/NPY to ethanol sensitivity and to stress
  • NPF levels are reduced by rejection and enhanced by mating
  • down regulation of NPF leads to increased drinking.
  • implant electrodes into mouse brain, it will press lever to get neural reward at expense of eating, drinking, sex.
  • use optogenetics to stimulate NPF neurons. Flies exhibit preference for part of chamber where they receive the stimulation.
  • can now map all reward systems using Gal4 brain lines and optogenetics.
  • use camera tracking to record 90-100 behavioral features of individual and individuals interaction with others. computer recognizes these behavioral types automated. Called JABBA (freely available)
  • rejected flies keep their distance from other flies. They also touch appendages less often
  • they tend to back away from other flies. Mated flies hang out in little hubs, and back up primarily to get out of the hub.
  • mix pairs of rejected and mated flies and both behave like the rejected.
  • do rejected males have eating disorders? -> No
  • Circadian effects? Yes also linked. chronic loss of sleeep?
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Fly Meeting, Saturday 3/7/15


Early zygotic dosage compensation in Drosophila melanogaster is Sxl dependent.

  • Susan E. Lott1,2, Jacqueline E. Villalta3, Michael B. Eisen2,3. 1) Evolution and Ecology, University of California, Davis, Davis, CA; 2) Department of Molecular and Cell Biology, University of California, Berkeley, CA; 3) Howard Hughes Medical Institute, University of California, Berkeley, CA.
  • Hox gene intro
  • DNA binding domains highly conserved. Yet still high specificity
  • Ubx activates svb responsible for producing trichomes
  • Ubx everywhere, get A1 everywhere (has tricomes)
  • ID enhancers (E3 and 7), both respond to Ubx the same way
  • No canonical binding sites for homeobox / Ubx.
  • chopped up enhancer and looked for which reions can bind ubx in vitro
  • find cluster of low affinity Ubx binding sites.
  • delete individual low affinity sits still see expression
  • delete 2 (or 3) binding sites, abrogate expression.
  • SELEX-seq — deep sequence screening for affinity range of TFs for all sequences.
  • (AbdA binds same sequence as Ubx)
  • Lowest affinity sites of the range that Ubx binds are the most Ubx specific.
  • increase binding strength, start getting more expression in AbdA region.
  • add high affinity binding sites more ectopic expression in anterior range, (less expression in native sites
  • Ubx hets in minus one binding site, no change in WT, but change in Ubx hets
  • low or high temp treatments also have stronger effects on site loss than wt at 25C

Low affinity binding site clusters confer Hox specificity and regulatory robustness.

  • Justin Crocker1, Namiko Abe2, Lucrezia Rinaldi2, Alistair P. McGregor3, Nicolás Frankel4, Shu Wang5, Ahmad Alsawadi6,7, Philippe Valenti6,7, Serge Plaza6,7, François Payre6,7, Richard S. Mann2, David L. Stern1. 1) HHMI Janelia, Ashburn, VA; 2) Columbia University Medical Center; 3) Oxford Brookes University; 4) Universidad de Buenos Aires; 5) New Jersey Neuroscience Institute; 6) Centre de Biologie du Développement; 7) CNRS.

A cell type specific transcriptional repressor directs selective upregulation of terminal differentiation program.

  • Jongmin Kim, Margaret Fuller. Stanford University, Stanford, CA.
  • testis zinc finger was necessary for proper differentiation in the testes
  • Made antibody, find protein is nuclear and only in differentiating cells not stem cells.
  • phenotype similar to tMAC, another TF which activates testes specific transcripts.
  • most genes strongly affected in tMAC not affected in tZNF
  • group of genes strongly upregulated in tZNF KO: gut and neuronal RNAs
  • tZnF is a repressor, tMAC activates a broad swath of genes, and tZnF keeps off a substantial fraction of these that are not wanted in testes.
  • maybe tMAC has promiscuous binding? (what conserves these sites?)

Genome-wide futile cycling by Hairy transcriptional repressor reveals mechanism for development of nascent gene regulatory networks.

  • Kurtulus Kok2, Ahmet Ay3, David Arnosti1,2. 1) Dept Biochem & Molec Biol, Michigan State Univ, East Lansing, MI; 2) Program in Genetics, Michigan State University, East Lansing, MI; 3) Departments of Biology and Mathematics, Colgate University, Hamilton NY.
  • “shotgun model of transcriptional regulation”
  • “Tijan era of precise TF binding is falling apart” – TFs bind where they are supposed to and where they are not supposed to.
  • Fisher et al 2012 — lower occupancy sites tend not to drive expression — truly background binding
  • early embryo: broad activators and specificity in short range repression.
  • Short range repressors and long range repressors
  • both short and long range interact with both Groucho and CtBP. Still a mystery.
  • Chromatin?
  • Hairy represses H4Ac across a long domain. Knirps has a very local effect on H4Ac
  • over-express Hairy throughout the genome.
  • more sites change than transcription change
  • Hairy modifies chromatin at some sites without changing transcription.
  • futile cycling? genome wide effects that may have no direct consequence?
  • Hairy may target silent genes it doesn’t care about.
  • How many active genes get Hairy binding when your over-express it?
  • Hairy does not have epigentic

Single base differences in a shared cis-regulatory element are critical for rhodopsin expression in distinct photoreceptor subtypes.

  • Jens Rister, Claude Desplan. Department of Biology, New York University, New York City, NY.
  • Broad ad restricted genes have a shared motif (called P3 or RCSI 11 bp)
  • trpl GFP observe reporter in all cells, mutate motif, lose expression.
  • also true for the motif in rhodopsin
  • restricted rhodopsin actually have varients of these motifs.
  • specific, subtle basepair changes completely define this specificity
  • Question: are these motifs multermized in the enhancers? Comment on the organization of these sites relative to the promoter?
  • change single basepair in the site and expand from subset of binding sites to all cells.
  • created a Pph13 site? Yes. Can mutate
  • see partial repeats upstream required for full expression.
  • combine two restricted sites — get no expresion

Integration of repressive and patterning inputs at cardiac gene loci.

  • Jemma Webber, (Rebay lab)
  • Yan and Pointed regulate genes downstream of RTK signalling in bistable way.
  • Yan (repressor) and pointed regulate muscle heart enhancer
  • Yan binds upstream of eve promoter to D1 enhancer (Webber 2013)
  • long range interactions occur betwen the HME and D1 (how do you distinguish these from enhancer promoter interactions)
  • Pnt and Yan have similar binding profiles at eve locus.
  • imaging shows Pnt and Yan colocalize in several of the mesoderm cells (though both have complex patterns)
  • sequential ChIP fails to detect co-occupancy
  • Pnt recruitment at D1 still occurs in Yan null
  • Yan and Pnt co-occupy D1 region with Gro.
  • Pnt is required for Gro recruitment (reduced in pnt null). Pnt may also be a repressor.

Differential binding and activation of enhancers by Bcd and Otd in the embryo.

  • Rhea Datta, Danyang Yu, Stephen Small
  • Bcd is missing from the genomes of most insects
  • Otd plays a Bcd like role in other insects
  • both K50 Homeodomain TFs TAATCc have same binding specificity
  • does Otd bind Bcd targets? Bcd ~3000 peaks, Otd only 500, so no:
  • Question: is the difference in Otd and Bcd binding concentration dependent?
  • in late embryo Otd binds amny of the regions (870) of the regions bound by Bcd early
  • Bcd and Otd bound to enhancers that are non-functional early.
  • how are late stage enhancers kept off at the early stage?
  • replace bcd with maternal gradient of Otd (otd/bcd swap):
  • does not result in rescue: looks like bcd mutant by in large
  • activates only a subset of Bcd target genes.
  • swap bcd homeodomain in Otd: get rescue. Vice versa, no rescue.
  • Use Bulyk micro-arrays to compare Bcd and Otd binding site strengths.
  • But: Otd preferred sites are enriched in Bcd bound regions.
  • Zelda and Hb sites are important
  • Add Zelda sites to late acting enhancers — some of these get activation
  • late responsive sites are enriched in tramtrack (maternally provided, rapidly degraded). Remove tramtrack repressor sites in late enhancer, get activation!
  • what fraction have tramtrack sites?

Transcriptional activation by a low-complexity domain is a conserved feature of Zelda and orthologous proteins.

  • Danielle Hamm, Eliana Bondra, Melissa Harrison. Dept. of Biomolecular Chemistry, University of Wisconsin, Madison, WI.
  • MZT and Zelda
  • how does Zelda mediate genome activation?
  • 4 zinc finger region forms the DNA binding domain, accounts for 100% of the footprint.
  • Mutation in any of zinc fingers abrogated transcription. Conclude all are necessary for binding.
  • There is a splice form variant that lacks 3 of these 4 (but has most of the other sequence), ZLD-PD. No transcription activation from Zld-PD
  • Zld-Pd reduces activitiy of Zld-PA (full-length)
  • N terminal domain still essential for activity. Fused this to Gal4-DB. test with UAS-firefly.
  • low complexity region 904-1300 has activator effect
  • this plus the DNA binding Zn-fingers are sufficient to activate transcription.
  • all zelda orthogos can activate (somewhat) transcription in D mel (out through Anaopholese)
  • alternative splicing could be used to stop zelda activity.

Discovery of Novel Enhancers Using Natural Variation. Ashley Jermusyk, Sarah Gharavi, Gregory Reeves. Chemical and Biomedical Engineering, North Carolina State University, Raleigh, NC.

  • using natural variation in DGRP lines to ID enhancers
  • Quantifying variation in Kr posterior border between lines
  • Question: have you tried hierarchical clustering on changes between lines? See if this clustering reflects correlations in mutations.
  • grouped SNPs into 100 bp to 1 kb test enhancers.
  • Question: did you swap back any of these SNPs?

Shadow enhancers enable Hunchback bifunctionality in the Drosophila embryo.

  • M. Staller, B. Vincent, M. Bragdon, J. Estrada, Z. Wunderlich, A. DePace. Systems Biol, Harvard Med Sch, Boston, MA.
  • eve 3/7 regulation
  • general approach: map TF concentration to mRNA concentration with linear functions.
  • Hb repressor model sufficient to explain 3/7 enhancer
  • eve 3/7 expand in snaE>Hb. need to invoke Hb as an activator on 3/7
  • How is Hb bifunctionality encoded in the genome?
  • eve 3/7 enhancer doesn’t bend like the wildtype 3/7 stripes. It retracts like the repressor model.
  • Kr proximal and distal respond differently.
  • proximal expands, distal retract
  • hunchback changes its promoter
  • is this extension in stripe 7 temporal specific?
  • Hb switches promoter (which may or may not change its sequence).

Changes in a P-MAD binding site underlie species diversity of wishful thinking patterning.

  • Rob Marmion1, Milica Jevtic2, George Pyrowolakis2, Nir Yakoby1. 1) Biology Department and Center for Computational and Integrative Biology, Rutgers University, Camden, NJ; 2) Institute for Biology I, Albert-Ludwigs University of Freiburg, Freiburg, Germany.
  • wishful thinking previously only implicated in axon guidance. Find a role in oogenesis.
  • dpp is anteriorly localized.
  • Wit is necessary for dpp signaling and its expression is regulated by the pathway.
  • promoter bashing: characterize enhancer of Wit
  • mutation of MAD sites disrupts reporter expression.
  • Wit is wider in virilis (4 appendages)
  • virilis enhancer drives wider expression in D mel (and patchy in posterior).

A genetic screen for new Polycomb group genes.

  • James Kennison, Monica Cooper. Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD.
  • Can we make a better screen than Sex combs?
  • PREs have pairing sensitive silencing. PRE in transgene, 30-50% of insertion sites the homozygote eye color gets lighter.
  • identified 5 PRE candidates that exhibit pairing sensitive silencing from scr. Focus on one from second intron of Scr. Biggest change from red to white
  • Use FLP/FRT to generate clones in the eye (eye-FLP)
  • mutagenize flies, screen for red conversion due to loss of pairing
  • 370 – 400 lines
  • 20% of mutants alter copy number of derepressed genes
    • derepression primary in posterior of eye, eye is rough / not dividing well. mutants survive as homozygotes.
    • Meiotic recombination strongly surpressed: can’t map and hint that something is wrong.
  • 51 complementation groups. 30 have multiple alleles. 14 of these correspond to known PcG. Get everyone except mxc.
  • do get extra sex combs from scr in new mutants. Need to map now.
  • For 3 genes with a single allele, there are two ORFs.
  • 22 of the genes map to a single transcription unit.
  • 15 chromatin factors: Spps, grh, Dsp1, ocm, (previously implicated)
  • also new ones: ftz-f1, Nf-YB, aminoacy-tRNA synthass and some protein kinases

Super-resolution imaging of chromatin nanostructure reveals tight coupling of epigenetic state and 3D genome organization.

  • Alistair Boettiger1, Bogdan Bintu2, Jeff Moffitt1, Brian Beliveau4, Chaoting Wu4, Xiaowei Zhuang1,2,3. 1) Chemistry and Chemical Biology, Harvard University, Cambridge, MA; 2) Department of Physics, Harvard University, Cambridge, MA; 3) Howard Hughes Medical Institute, Harvard University, Cambridge, MA; 4) Department of Genetics, Harvard Medical School, Boston, MA.

The RNA binding protein Arrest (Aret) regulates myofibril maturation in Drosophila flight muscle.

  • M. Spletter1, C. Barz1, A. Yeroslaviz1, C. Schönbauer1, D. Gerlach3, I. Ferreira1, M. Sarov4, A. Stark2, B. Habermann1, F. Schnorrer1. 1) Max Planck Institute for Biochemistry, Martinsried, Germany; 2) Research Institute of Molecular Pathology, Vienna, Austria; 3) Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; 4) Max-Planck-Institute of Cell Biology and Genetics, Dresden, Germany.
  • fast contracting flight muscles have fibular structure
  • all other muscles have tubular organization. How do these differences arise?
  • spalt major is a master regulator of fibular muscle (KO -> convert to tubular muscle).
  • mRNA seq in spalt KO
  • RNA binding protein Arrest is regulated by spalt. Arrest KO muscles pull themselves apart.
  • Arret is expressed in cyto and nuclei early in development and in immature fibrils. later it moves into the nucleus. (72 hours)
  • Sarcomeres widen (factor of 1.5) in WT. This widening growth fails in arret KD
  • seq Arret KO, 90% of exons change — splicing effect more than tx effect
  • Aret promotes the inclusion of fibrilliar exons, inhibits the inclusion of tubular exons.
  • Arrest phenotype looks like classic myosin over-activity defect (muscles over contract and snap)
  • myosin mutant blocks this pull the muscle apart phenotype.
  • Strn is one of the differentiatially spliced genes.

Ultraconserved core elements are an essential feature of insect enhancers.

  • Thomas Brody, Ward Odenwald. Neural Cell-Fate Determinants Section, NINDS/NIH, Bethesda, MD.
  • late neuroblact ultraconserved enhancer
  • enhancer consists of clusters of conserved octomer bindings sites (repeat sequences)
  • conservation of mouse to lamprey (2.5 By) including insertion deletion lack.
  • (260 My) Anapholeses to D mel.

Saturday evening talks

Preventing age-related metaplasia promotes homeostasis of the gastrointestinal tract and extends lifespan.

  • Hongjie Li1,2, Yanyan Qi1, Heinrich Jasper1 1) Buck Institute for Research on Aging, Novato, CA; 2) University of Rochester, Biology Department, Rochester, NY.
  • up-regulation of Jak-STAT pathway leads to gut degeneration
  • inhibition of Jak-STAT matains gut homeostasis and extends lifetime.

Regulation of metabolism and insulin sensitivity by Sir2 in Drosophila.

  • Rebecca A. S. Palu, Carl S. Thummel. Human Genetics, University of Utah School of Medicine, Salt Lake City, UT.
  • Sir2 (ideintified in yeast associated with aging) is a metabolic and epigenetic regulator
  • involved in a variety of metabolic processes. Details remain unclear (aging, diabetes, Obesity etc)
  • sir2 mutants become increasingly insulin resistant with age of animal
  • old sir2 mutants clear glucose much more slowly than wt or young sir2 mutants
  • hyperglycemia sets in early. then insulin resistant obesity sets in. then glucose intolerance (about a week apart each).
  • RNA seq mutants at 2 week time point.
  • strong degree of overlap between genes regulated by sir2 and genes regulated by hnf4 and foxo (known targets of sir2 in mammals. invovled in glucose matabolism)
  • Propose Sir2 de-acytelates these TFs to change gene expression to change metabolism in response to nutritional cues.’

Systemic organ wasting induced by localized expression of the secreted insulin/IGF antagonist Imp L2.

  • Young Kwon1, Wei Song1, Ilia Droujinine1, Yanhui Hu1, John Asara3,4, Norbert Perrimon1,2.
  • Wasting: process of losing mass (opposite of growth)
  • systemic organ wasting associated with extreme starvation, infection, severe infection, cancer and other diseases.
  • cancer cahexia — cancer associated wasting
  • Hippo signaling is a master regulator of growth via Yki activation, leads to localized cell proliferation.
  • Fly phenotype: ovary atrophy, fat body disappears, bloated, translucent abdomen.
  • Impl2 RNAi rescues the systemic organ wasting phenotypes

Mechanism of Body Fat Regulation by Split ends.

  • Kelsey Jensen, Tânia Reis. Department of Medicine, University of Colorado Medical School, Aurora, CO.
  • use sucrose PBS solution to screen float vs sinking L3 larvae to ID fat regulation
  • (screen performed by Reis in Hariharan lab in 2010)
  • trying to find new genes that regulate fat
  • use carnegie FlyTrap project to screen 33 lines which have GFP. 18 of which are expressed in the fat body
  • next step: knock these genes own specifically in the larval fat body.
  • gene split ends (conserved between flies and mammals) not previously linked to metabolism.
  • over-expressed copy of gene leads to high density low fat larvae
  • Is there a behavioral / eating / moving change?
  • KD larvae over-eat (may contribute to being fat).
  • overexpression larvare eat normally.
  • Film movement: both over-expression and lean larvae move slower than their controls.
  • fat larvae actually starve first (opposite to expectation). Can’t utilize their fat properly? perhaps this leads to the over-eating phenotype?
  • increase in fat metabolism, decrease in sugar metabolism
  • Split ends (Spen)
  • now testing expression levels of homolog in mice (MINT). different levels of Spen in fat liver than in lean liver.

Saturday Evening Session

Angela DePace

  • cooperativity mostly thought about in terms of pairwise interactions
  • long distance cooperativity?
  • ‘linear framework': Markov chain transition between microstates
  • ‘Ahsendorf et al’ BMC Biology 2014
  • need more than pairwise cooperativities to get hill like functions
  • asymptotically approach hill coefficient equal to N sites
  • doesn’t really matter if assume all bound yields transcription or any bound yield transcription.
  • away from equilibrium more easily get sharp switching.

Michael White

  • Goal: quantitative relationship between sequence and gene expression
  • Hh signaling makes Ci an activator. Otherwise it is a repressor
  • Gene expression is proportional to occupancy
  • in middle of gradient, activation wins at low affinity and repressor wins at high affinity, (because repressor is more cooperative and switches sharper).
  • 3-strong sites, repressed. (cooperative binding dependent?). delete 2 of the activator sites, now it’s an activator.

Golding: Quantifying the stochastic kinetics of Gene regulation

  • Heng Xu and Anna Sokac
  • How does binding of TFs drive stochastic process of mRNA production?
  • how do multiple TFs regulate a single gene?
  • Progress:
    • Measure concentration of a given TF factor
    • promoter activity
    • TF binding
  • in individual nuclei of WT embryos
  • use stochastic theoretical analysis to connect this to binding
  • have the endogenous species (check)
  • have ‘absolute numbers’ (maybe….)
  • can we interpret the full dataset rather than averaging over nuclei?
  • 2 step promoter model
  • Kon is Bcd dependent. Describe that as the Hill function of Bcd function.
  • signal to noise is around 1%. But pool many nuclei and this may be enough
  • linear proportionality to 3 loci measured by ChIP (but I don’t believe quantitative ChIP data either)
  • cycle 12 embryos, see a double plateau of bcd concentration at the transcription site as a function of AP position.
  • propose Hb binding represses hb.
  • Two color control?


  • triplet code
  • Cis-regulatory code: on in correct cell type, off in correct cell type.
  • need inputs, outputs, and a way to compare them.
  • need to work at cellular resolution.
  • will discuss control of the even-skipped
  • need to rely on some phenomenology (careful guessing). Function tends to reside in multiple sites. Can get substantial amounts of synergy.
  • Regulatory mechanisms considered: protein binding DNA steric competition, cooperative binding, short range repression by quenching (150 bp) phenomelogical
  • coactivation (150 bp) phenomological
  • activation by arrhenius rate law.
  • PWMs are determined independently
  • 3-7 + 2 fusion
  • sepsid stripe 2 works in D mel (‘shit eating flies’) Homology in stripe 2 is extremely weak (2 binding sites or so). Predict that sepsid stripe 2 will be posteriorly shifted.
  • most activation in stripe 2 was coming from co-activated Hb sites
  • Hb-3 amplifies signal but does not give expression by itself.
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Fly Meeting, Friday 3/6/15


Giaovoni Bosco

  • in wild 80% of fly larvae of been injected by wasps
  • Q: is there wasp/wasp competition inside the same chamber
  • Flies produce 80% fewer eggs (apoptosis in ovary) when exposed to wasp.
  • in separate chambers in fly condos, observe reduced egg laying in unexposed neighboring combos
  • add GFP flies (naive) to exposed wt flies, see reduced egg laying in FGP
  • how long does the repressed egg laying state last?
  • physiological change in ovary also observed in GFP flies
  • memory returns to normal over 240 hours (somewhat gradually)
  • Orb2 mutants go back to normal egg laying within 24 hours (gene involved in memory)
  • rut1 and Orb2 mutants cannot teach His-GFP flies to repress egg laying
  • ninaB (vision) is required for response to wasp. See no effect in the dark or with blind flies.
  • Learning also requires vision (Orb2, Adf1, dnc, rut required). Memory required for sustained oviposition.
  • Requires 2 wings to teach. Only females teach. Males don’t help. Not auditory (can block auditory and olfactory)
  • Compensentory increase in life span? (Otherwise what’s the adventage of decreasing your fitness, even in presence of the predator)
  • In wild they seek out high ethanol food when egg depsition is low, and then it ramps up.
  • Is it parasytic wasp specific? what about other wasps? Yes. Only respond to parasytic female wasps.

Charaterization of a long-distance neurotransmitter recycling pathway essential for Drosophila visual transmission. Ratna Chaturvedi, Hong-Sheng Li. Department of Neurobiology , University of Massachusetts Medical School, Worcester, MA

  • local synaptic vesicle recycling can create interference
  • long distance recycling (distal from the synapse) can avoid this.
  • Does it occur in the fly?
  • Histamine (analog of glutamate signaling in fly vision) metabolites are present in the glia cells (not just the neurons) suggests there is a longer range recycling.
  • block gap junctions, see reduction in histamine in glial cells.
  • these flies are also deffective in synaptic transmission.
  • don’t know transporter (what was that about the gap junctions?)
  • RNAi screen knockdown of BTA see reduced photo-signal transition. (Blind not lethal).
  • only neurons express BTA, not Glia.
  • only photoreceptor neurons damaged.
  • Deleted BTA with CRISPR, acAGATACA motif validated RNAi
  • BTA deletion flies accumulate carcinine in the lamina glia, and is not recycled back to the photoreceptors.
  • use moving block ‘threat’ to assay visual responsiveness. BTA nulls blind.

Contribution of sex, genotype, and environment to individual gene expression profiles. Kseniya Golovnina1, Yanzhu Lin2, Zhenxia Chen1, Haiwang Yang1, Hina Sultana1, Brian Oliver1, Susan Harbison2. 1) NIH/NIDDK, Bethesda, MD; 2) NIH/NHLBI, Bethesda, MD.

  • DGRP lines
  • high variability even when same environment, sex, and genotype.
  • How variable is gene expression among individual flies?
  • 16 genotypes, 3 different environments
  • 8 females, 8 males, do RNA-seq (768 libraries)
  • Of 15695 genes, 95% differentially expressed due to sex
  • 31% of genes with unknown function show sex specific expression differences. More often male specific up-regulation.
  • Male expression values more variable than females
  • Q: do you see a correlation in variabilty to being on the X chromosome?
  • ID genes more sensitive to environment.
  • find examples of variance explained by sex-environment / sex-genome co-variation.
  • How was development stage controlled? : just age
  • What was the environment variation: not stated.
  • Variation in expression correlated to anything? When we look across all genes

The evolution of maternal mRNA deposition and zygotic genome activation across 14 Drosophila species. Joel Atallah, Susan E. Lott. Evolution & Ecology, University of California – Davis, Davis, CA.

  • generated 144 libraries (14 species, 2 stages) look at zygotic genome activation
  • look at divergence in zygotic / maternal FPK ratio for genes. Take correlated genes from mel and compare out to other species — correlation drops (not too surprising. but I suppose which gene change will be interesting)
  • Look at maternal only, zygotic only, and both
  • most transitions are the both to maternal or the both to zygotic
  • very few examples in evolution of zygotic to maternal or maternal to zygotic: changes pass through an intermediate. (again, what you would likely guess)
  • CAGTAG / TAGTeam sites are enriched in the promoters of genes that become zygotic / are zygotic. (Motif is well conserved. Maybe an 8mer instead of 7mer in pseudo).
  • find motifs that are lineage specific

Three-step mechanism for the spatial and cell-cycle dynamics of pericentric chromatin. Eric Joyce, Tharanga Senaratne, Ting Wu. Department of Genetics, Harvard Medical School, Boston, MA.

  • screen for genes that affect pairing of peri-centric chromatin
  • ID condensin II components, centromere components
  • clustering is cell cycle dependent: stain CycB, more clustered in G1. (cycB is on in G1 only (?))
  • centromeres get clustered in telophase
  • knockdown centrosomes — still get division, centromeres don’t cluster in mitosis, remain unclustered in G1
  • centromeres do not have an intrinsic ability to find eachother
  • (Does this extend to pairing? of other loci?)
  • Mcph1 RNAi, heterochormatin becomes disperse. No visible H3K9me2 when heterochromatin is disperse. Some repetive regions start getting expressed.
  • centromeres redistribute in a condensin II dependent manner.

Mrg15-dependent binding of Cap-H2 to chromatin is required for chromosome organization and regulation of gene expression. Heather Wallace, Huy Nguyen … Giovanni Bosco.

  • Condensin II drives axial compaction and un-pairing
  • Mrg15 chromodomain protein interacts with condensin II (H3K36me3 interacting)
  • required to mediate shortening and unpairing.
  • Mrg15 binding sequence is required for its interaction with CapH2 (perhaps it targets CAP)
  • FISH probes on chromosome measure compaction, (overexpress?) CapH2 GFP, chromosome shortens (distance > 2 um to closer than 2 um)
  • ChIP-seq shows very high correspondence, both at active locations
  • most of the same genes (95%) respond the same in RNAi knockdown of Mrg15 vs. Cap-H2 (same ~3400)
  • most genes downregulated on loss of CapH2

Trimethylation of Histone H3 at lysine 27 by Polycomb Repressive Complex 2 and its role in epigenetic memory. Rory T. Coleman, Gary Struhl. Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY

  • Propose H3K27me3 carries the memory across cell-cycles
  • DNA replication poses a challenge to histone-based memory (add unmodified histones)
  • generate Ubx-LacZ PRE mini-gene with a FLP-outable Ubx-PRE (also containing GFP in FLP-out)
  • look in wing disk where Ubx is silent. induce Pre excision,
  • cells remember off state for 12 to 72 hours — location dependent (nodum remains repressed for 72 hrs) mostly de-repressed by 110.
  • Only lose the memory of the off-state in actively dividing cells. G0 cells remain silenced.
  • expect dilution of K27me3 through progressive rounds of cell division.
  • after 12 hrs, drop to 32%, thereafter to 24% to 16% of H3K27me3. Rate of dilution is slower than predicted (7 cells divisions)
  • Have you done ChIP for PRC2? ( propose ‘free ‘ PRC2 passes on the mark.) — PRC2 components only ChIP to the PRE. Don’t see near by.
  • knockdown PRC1, lose repression while maintaining the H3K27me3, so K27me3 might be inheritable but is not intrinsically sufficiently repressive.
  • Q: looks like a potentially great system to study the relative roles of PRC2 and PRC1.

Tip60 HAT Action in Environmental Enrichment induced Cognitive Restoration. Songjun Xu. Biology, Drexel University, Philadelphia, PA.

  • Tip60 is an Alzheimer’s associated HAT
  • Tip60 is required to induce neuroadaptive transcriptional response to environmental enrichment (EE). what is EE?
  • 220 EE responsive genes, 43 sensitive to HAT,
  • TIP60 rescues E induction of synaptic markers in the AD neurodegenerative fly brain (when was the AD fly model introduced?)

Dietary restriction reduces transposable element expression in aging Drosophila heads. Jason G. Wood … Stephen L. Helfand, Brown University

  • How does chromatin change in age?
  • in yeast, normally repressed loci (mating loci, telomere) become expressed with age.
  • focus on repressive heterchromatin H3K9me3.
  • a lot of the heterochromatin we don’t have assembled very well.
  • with age, enrichment of H3K9me3 at centromeres decreases substantially (and chr4)
  • Hp1 follows
  • use LacZ moved into heterochromatin region, see increased expression in age.
  • low calorie fed flies lose silencing later.
  • how do native heterochromatin genes change expression with age? Some increase substantially with age (none decrease?)
  • in calorie restriction less increase in expression (most decrease)
  • Transposons: 2 types: cut and paste. Also retrotransposons, transcribe and jup. Lots of transposons. Pericentric region is gene poor but retro-transposon rich.
  • transcriptional and post-transcriptional silencing mechanisms combat retro-transposons.
  • rRNA depletion generally not designed for fly. Nugen custom depletion kit works better. TEs are repetitive, hard to align and quantify expression.
  • used RepEntrich: assemble all elements into a single ‘gene’. (not at all clear how this works).
  • Do any genes go down?

A novel chromatin factor Enhancer of Polycomb acts in somatic cells to maintain germ cell identity and activity in Drosophila adult testis. Lijuan Feng, Zhen Shi, Xin Chen. Biology, Johns Hopkins University, Baltimore, MD.

  • Approach: knockdown E(Pc) and exmaine effect in germ-cell lineage
  • compromised E(Pc) see extra mitotic division (overgrowth?)
  • may divide unsynchronously (not all remain connected by fusome, EdU variation).
  • E(Pc) enriched in promoter region of target genes that change in E(Pc) mutant, including Zfh-1.
  • overexpression of Yan componsates in part for E(Pc) knockdown.
  • E(Pc) is a component of Tip60.

Impacts of centromere misregulation on genome stability and cancer progression in a Drosophila model of glioblastoma. Nicole Beier, Renee Read, Gary Karpen

  • many centromere kinetochore genes are overexpressed in cancers. e.g. CENPA and HJURP
  • high levels of these proteins correlate to poor prognosis and susceptibility to radiation treatment.
  • CID overexpression causes genome instability
  • 70 fold overexpression find it throughout the genome. Leads to anneploidly
  • Find increased breaks (with chromosome arm BAC paints)
  • overexpress CID see fewer glia (kill cells)
  • overexpress CAL1 (chaperone assembly factor for CID), no real change
  • overexpress PI3K and CID get overgrowth (alone neither gives overgrowth)
  • CID + CAL1 see decreased proliferation (opposite of expectation)
  • endogenous CID is only enriched in centromere
  • 2x expression see increase at promoters of transcribed genes
  • expands into the gene-body on 70x expression.

A genome-wide resource for the analysis of gene function and protein localization in Drosophila.

  • Mihail Sarov1, Christiane Barz2, Katja Finkl2, Marco Hein2, Stephan Janosch1, Nicole Plewka2, Bettina Stender2, Dana Suchold1, Vinay Vikas3, Matthias Mann2, Mani Ramaswami4, K. VijayRaghavan3, Pavel Tomancak1, Frank Schnorrer.
  • 10,000 clones within their natural genomic environment
  • goal: generate multifunction tags (super-folded GFP) good for spatial organization, protein purfication etc.
  • ~12,000 genes cover by fosmids. ~11,000
  • inserted onto third chromsome. 800 genes tagged.
  • Do the proteins rescue? 15 don’t (including snail esg, and eya) TFs less likely to rescue.

Computational tissue labeling: Tissue and Cellular Recognition in Developing Drosophila Embryos. Soile V. E. Keranen

  • older embryos have variable size nuclei
  • two step segmentation: first isolate tissues and then cells
  • one approach: hand annotation in every third slice.
  • alternatively label tissue by gene expression.
  • however 70+ tissue and sub-cell types.
  • problem: only 3-5 colors. (sounds like an excellent system for MMECFISH
  • get tissue by nuclei/DNA patterns
  • then get nuclei. Use tissue specific nuclear parameters
  • ‘Fly Annotator’ program (looks like Matlab)
  • variable size and nuclear number between embryos (is this a staging issue?)
  • compare left-and-right paired organisms (like salvary glands). Left salvary gland is longer, left anal pad is longer
  • Training computer to automatically age rank embryos
    • overlay gut point clouds from embryos of different ages
  • pyramid contex features allow identification of organs
  • All 2-photon imaging.

Model-driven data visualization and quantitative animation of developmental signaling. Bomyi Lim (Stas Shvartsman lab)

  • with fixed tissue temporal staging is difficult
  • live imaging not available for many things (like dpERK)
  • want to combine different stains
  • use membrane length in blastoderm
  • use PCA in gastrulation. 5 components do a pretty good job
  • use a linear regression with 5 projection coefficients to
  • Q: If you apply the method to snapshots from live movies of different embryos, can you measure temporal variability in coordination of growth.
  • ERK is activated only in cells where rho was expressed (spitz must be short range).
  • add ERK from model and Ind from in situ stain to histone-RFP movie to create 3 color movie of ERK.

High-throughput Investigation of Drosophila Brains via Structure Based Similarity. Florian Ganglberge

  • 3d confocal stacks of 1000s of Gal4 driver lines expressed in the brain.
  • want to annotate neurons.
  • challenges: variable intensity, registration errors, background from other neurons labeled.
  • approach: ‘slow bulky Matlab script’. few hours for 1 search
  • optimized the method by downscaling image resolution, sparsification: remove noise and background.
  • added tool to 7x faster
  • can highlight the neuron in one image and find all the gal drivers that have that neuron ON in the database of images.
  • structure based queries work better than direct overlap (direct overlap is low).

An automated image analysis tool to track cell divisions during Drosophila axis elongation.

  • Michael F. Z. Wang1, Rodrigo Fernandez-Gonzalez1,2,3. 1) Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada; 2) Cell and Systems Biology, University of Toronto, Toronto, Canada; 3) Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Canada.
  • convergent extension intro
  • cell division is resolved along AP axis, contributing to elongation.
  • explain watershed algorithm (almost identical to matlab tutorial):
  • Threshold, dilation, inversion, distance transform, now watershed.
  • temporal alignment using cross correlation in paired binned images.
  • use ratio of perimeter to area (relative to circle) – this dereases and then increases in preparation for division
  • the ratio of the major and minor axes also helps ID pinching off cleavage furrow.
  • can now measure cell division orientation quantitatively for lots of cells. (compared to manual annotation).
  • look at the role of tension in the A-P resolution of division.

FlyVar: a database for genetic variation in Drosophila melanogaster.

  • Rui Chen1,2, Lichu Jiang1, Yong Chen4, Nele Haelterman3, Hugo Bellen2,3,5, Fei Wang4. 1) HGSC,Baylor College of Medicine, Houston, TX; 2) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas; 3) Program of Developmental Biology, Baylor College of Medicine, Houston, Texas; 4) Information Processing, Department of Computer Science and Technology, Fudan University, Shanghai, China; 5) Howard Hughes Medical Institute.
  • genome sequencing cost still dropping fast.
  • bottleneck is now to ID bases that change
  • high degree of variation in drosophila: 1 variation per 500 bp
  • don’t have genome of our earlier lab strains
  • lots of fly lines / mutants!
  • Approach to find the good stuff: remove parental varients. remove benign variants, remove unrelated genes, add additional specific filters.
  • FlyVar documents the benign polymorophisms.
  • If you know locus and you sequence it, you may find mutliple SNPs. Tool can help ID which were meaningful?

Identification of novel drug targets for Tuberous Sclerosis Complex by cross-species synthetic screens combining CRISPR based knockouts with RNAi. Benjamin E. Housden,

  • Tsc complex (mutated in rare benign cancer — simplest cancer genetics)
  • theraputic strategy is weak: inhibits overactivated mTor.
  • synthetic lethal screen: kill cells only which house the mutation.
  • e.g. Ben Haley’s double hairpin approach
  • why not use combinitorial RNAi?
    • incomplete delivery
    • incomplete knockdown
    • off target effects
    • limitations are compounded in combinitorial screens
  • use single RNAi screens in CRISPR generated mutant cell lines
  • goal: generate homogeneous cell cultures (challenging in tetraploid fly cells)
  • CRISPR with GFP co-transfection.
  • Drosophila cells don’t like to grow alone. They like colonies.
  • change culture conditions to survive sort, and 16% grow from single cell into full culture. Nice, Ajaz should talk to these guys
  • can create homozygous mutant lines. e.g. for TCS1 and TSC2
  • mutants keep on growing without serum as expected, are larger as expected.
  • RNAi screen all phosphotases and kinases in the genome.
  • only explore RNAi that have no effect on WT
  • synthetic lethal effects conserved through human cells.
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Fly Meeting, Thur 3/5/14

Thursday Morning Platform Presentations

Yohnns Bellaiche: cell division in morphogenesis

  • Intro: beautiful movie of zebrafish morphogenesis
  • system: Pupal morphogenesis
  • Goal: multiscale imaging –
    • 900 63x images (cellular resolution of whole tissue).
    • all 900 images every 5 min
  • Can segment and color-code cells, for example by size.
  • 50,000 cells, most junctions formed by cell division, formation of a single large interface.
  • important for polarity maintenance, lies behind clonal patch.
  • in cell culture, get actin ring that contracts to a point (not like long interphase in epithelial tissue)
  • Neighboring cell forces are necessary to get a long interface (oblate cells, get constriction to a point contact).
  • Two membranes invaginate to form long pairs of branches,
    • move over, myosin not must localized at interphase of cell, but also expressed inside cell edge of neighboring cell.
    • this is a response to the mechanical stress
    • this myosin helps hold the two membranes together so it invaginates as a narrow pennisula and not a big v (can laser ablate just the myosin in this cell and they spring apart into a v, but then recover).
  • MyoII in neighboring cell is therefore required.
  • Does membrane of neighboring cell get inserted between dividing cells?
    • Yes it does ingress!
    • but it retracts / degrades in the end.
    • F actin is recruited to the interface at the time of retraction of inserted membrane.
  • Prevent F-actin function, it is the neighboring cells membrane that remains and two cells separate.
  • Actin flow inside dividing cells drives withdrawl of the membrane
  • Quantifying cell behaviors: size/shape change, cell rearrangememnt, cell division, and delamination. Plot spatial frequency of these events throughout the tissue.
  • estimate junction stress from lattice image.

Genetic Conflicts During Meiosis Drive Innovation in Centromeric Proteins — Harmit Malik,

  • Adapting to a changing / co-evolving environment
  • genetic conflicts within the same genome
  • conflict between ‘selfish centromeres’ and chromosome segregation?
  • Male meiosis in plants and animals
    • one homolog can poison the other sibling chromosome sperm
    • highly adventageous to the one chromosome that will win
    • disadventageous as fertility drops 50%
  • female meiosis (in plants and animals), only 1 cell wins the preferred position
    • if one chromosome can orient itself better (faster microtubule recruitment) can win more often. Selfish centromeres can increase transmission rate past 50%
    • examples: Robertsonian chromosomes (fusions of centromere-at-end chromosomes) These are normal, our chr2 is a Robersonian fusion still separate in great apes.
    • over-recruitment of centromeric proteins.
    • in male meiosis suffer fertility (male meiosis ‘likes symmetry’) (.1% of humans are Robertsonian)
  • 60:40 segregation will sweep Drosophila in 10,000 years
  • In Drosophila centromere boundaries / strengths are set up epigenetically
  • heterochromatin protein over expression can encroach on centromere and vice versa.
  • selfish centromeres try to get larger, penality paid in the males. intense evolution at centromere and centromeric proteins.
  • Non-canonical nuclesome (H3 replaced by CENP-A) in centromeres
  • swap Cid (fly chromosome centromeres) between simulans and yakuba, viability 95% (in both male and female F1) (viability not fertility?)
  • predict reconstruction of ancesetoral Cid, no effect on F1 female viability but strong effect still in male.
  • HP1b non-essential, but its daughter gene Umbrea does localize to centromere and is essential for normal cell division. Knockdown results in massive aneploidy.
  • a constant turnover of essential centromeric proteins.
  • C elegans ar holocentric (whole chromosome is a centromere, rather than concentrated at point on chromosome).
  • Lepidoptera, Hemioptera, all lose CenH3 and have holocentric chromosome division.
  • loss is specific and multiple times in the insect lineages.
  • old genes can become dispensable and young genes essential.

Angela Stathopolous: mesoderm cell migration


  • FGF ligands pyramus and Ths expressed in different tissues
  • FGF signaling required for proper mesoderm spreading (should form a nice internal monolayer)
  • cells don’t cross the midline
  • in htl mutants, upper domain cells do not exhibit directed movement and cross back and forth across the midline.
  • Role of leader cells — compotent to move in mutants or wt. FGF not so much chemo-attractant. More important in ensuring symmetric collapse of the mesoderm.
  • Conducted genetic screen (311 UAS lines), found 10 genes that affect mesoderm spreading.

Screen results:

  • Trol (HSPG) is required for furrow collapse
  • Sdc is required for intercalation and differentiation.
  • ectopic expression of pyr or ths in mesoderm leads to lumpy pile up of cells in mesoderm.
  • E-cadherin (normally down regulated in mesoderm). knockdown or overexpress lose monolayer of mesoderm.
  • E cad localization changed in htl mutants.
  • propose snail is effected by FGF. sna up-regulated in absense of FGF, down-regulated by FGF.
  • propose FGF regulates spreading by controlling adhesion state through E-cad regulation.

CVM cell migration

  • long range 6 hour migration
  • cells express heartless. Follow a track of cells expressing Pyr and Ths.
  • In FGF mutants cells still migrate, but they intermix
  • why do leader cells move faster? posterior cells hardly move…
  • FACs to isolate CVM cells and RNAseq
  • find enrichment in lipid synthesis in CVMs.
  • sphingolipids regulate germ cell migration. watch comigration of germ cells and CVMs.
  • lipid mutants show defects in migration.

Stas Shvartsman


  • ERK signaling – used multiple times in development
  • excessive ERK activation lead to many varieites of prevelant developmental abnormaliites.
  • localized RTK responds to transient pulses of ERK throughout development in many tissues.
  • ERK activates ind
  • will describe mathematical model that captures behavior semi-quantitatively and makes several clear predictions.
  • ### reconstructing dynamics
  • don’t have a live sensor
  • compare stills to database of live imaging movies to match morphology. record movies in the same vertically orienting PDMS device.
  • linear regression model matches features of images (5 dimension 5 component PCA mapping). Accuracy is better than 3 min (presumably matching embryo to embryo)
  • Question: does the 3 min accuracy come from intrinsic embryo to embryo variation, or uncertainty in the morphogenic mapping

Ind activation

  • receptor uniformly expressed, activated by Vn and Spitz. Vein is dispensible, Spitz (regulated by rho) is essential – necessary and sufficient. Don’t see any consequence from removing vn.
  • ERK signal grows as the t^3 over time.
  • Pathway: Zld activated EGFR, Star and Spitz. Ligand grows linearly in time, receptor grows linear in time, integrated this rate over time, expect cubic growth.
  • Ind is expressed when ERK crosses a basic threshold.
  • delay induction in csw mutant, lower levels of ERK, takes longer to cross threshold, still turn on Ind.
  • How is ind responding to ERK? – regulated by Cic. ERK relieves Cic repression.
  • ERK first inactivates Cic, which is then exported and degraded.


  • upstream factors of ERK frequently mutated in developmental defects and cancer
  • propose that cancer assoicated mutations will be lethal in development
  • introduce mutations from human abnormalities into fly embryo.
  • change ERK pulse. Both developmental abnormality and cancer mutations cause strong lethal phenotypes in fly.
  • also developing computational tools.
  • reduce Cic, also (reduce) ERK signalling

Maria Dominguez: Addressing complixity in Notch signaling and cancer


  • finding out the unknown unknowns of cancer
  • ‘peto’s paradox': no relationship over species between cancer and organism. Mice have higher incidence of cancer than humans, whales have less.
  • No / almost never cancer in naked mole rate. — lower metabolism, less Reactive Oxygen species
  • Tumor cell communication is important.
  • Nutrition is relevant.
  • Size regulation / growth regulation / when to stop is relevant. — best understood in flies (?) Notch povides growth directions

Notch-Pten/Akt in cancer

  • Delta upregulation minor overgrowth
  • Delta + ATK upreguatlion dramatic cancer (clearly visible in eye tumor)
  • ATK is downstream of INLS/IGF/RTK/PI3K and upstream of dTOR
  • PTEN links NICD to Atk1
  • Screened sigma-aldrich library of pharmological active compounds looking for effectors of (PTEN?)
  • 150 flies screened per each 1000 compounds, ID 62 compounds that attenuate and 58 that enhance eye tumors
  • 23 of the 62 reduce growth in human cultured tumor cells. ~6 of these don’t affect growth of non-tumor cells. (what happened to PTEN / NOTCH?)
  • chose drug that has the strongest effect at lowest concentration.
  • find atp syn Beta gene
  • Warburg hypothesis: mitochondria dysfunction causes tumors.
  • Test: overexpress Delta and knockdwon atp synthase beta -> tumors
  • mitochondria dysfunction facilitates tumor growth through Delta/Notch signaling (these dysfunctional cells should die.
  • Delta+ and PTEN-kd flies have elevated levels of ROS (using ROS Gstd1-GFP sensor).
  • chemotherapy elevates ROS to kill tumor cells.

Juergen Knoblich, (IMBA) Stem Cells


  • stem cell number rapidly decreases during development.
  • Timely loss of stem cells is important to avoid child tumors
  • Drosophila neuroblasts (neural stem cells). Responsible for generating all the neurons and glia in adult brain.
    • Type I: dived assymetrically, differentiatin cell divides into 2 neurons. (mice)
    • Type 2 proliferative cell is generated from first asymetric division and then divide and differentiate. (flies and humans)

Drosophila neuroblasts

  • Drosophila pupal NBs shrink before disappearing (in diameter before differentiating),
    • consequence of metabolic limits of non-eating pupae?
    • Follow neuroblast division in culture to test.
    • due larval and pupal NBs culture differently? Pupal neuroblasts become smaller with cell divsion, larval ones do not. Not related to division rate.
  • known growth pathways do not appear to be involved. — Genetic screen:
  • express luciferase in neuroblasts.
  • Find 300 RNAi lines that affect behavior, none appear to be off-targets
  • 8 of these map to Mediator complex.
  • knockdown mediator components (Med27, Med10), get cell-type specific effect: more neuroblasts, less shrinkage and death. Increased diameter of neuroblasts.
  • Question: Does it matter which subunits of mediator you knockdown? (Is mediator functioning in a modular way?)’
  • Mediator is required for Ecdysone response (major hormone that triggers instar changes, spikes in pupation.
  • Mediator regulates metabolism.
  • NB cell cycle exit requires alpha-KGDHC (knock this down and they don’t exit / differentiate). (Gate keeper of Krebs Cycle, catalyzes the first irreversible step of Krebs. Generates NADH for respitory chain.
  • Mediator is required for upregulation of oxidative phosphorylation regulation.
  • Proliferative tissues (staying same size) don’t do Krebs, rely on anaerobic metabolism.
  • Metabolism change effects change in cell fate (rather than v.v.)

Thursday Evening Session: Organogenesis and gametogenesis

Weischaus lab Nucleolus formation

  • rRNA synthesis starts by cc11. Nucleolus doesn’t form until cc13.
  • phase separation model
  • RNA PolI and fibriliarin form in the absence of rDNA (but are much weaker).
  • rDNA is a nucleation site for fibrilian and PolI. Sites are less stable without it.
  • What explains the difference in intensity? If it is just a seeding effect, one might expect it is just time.

Yiqin Ma (Buttitta Lab, U Michigan): Examining chromatin structure in G0

  • Multiple types of G0: Reversible-quiescence, terminal differentiation, and senescence.
  • will study terminal differention G0 in fly wing (naturally synchronized)
  • exit cell cycle for last time to G0 24 hrs after pupal formation.
  • Flexible G0 phase 24 hrs to 36 hr (can still exit). Thereafter robust G0.
  • PcG /PRC2 and H3K9 methyltransferases up regulated on G0 exit
  • heterochromatin histones and proteins are disrupted (based on immune staining)
  • heterochromatin modifications are tightly associated with cell cycling status.
  • K27me3 and HP1 not required for cell cycle exit.
  • screen for other regulators,

the chromatin modifier trithorax regulates systemic signaling during imaginal disk regeneration

  • use UAS GAL4 to drive reaper under TS-Ga80
  • HS induce ablation (vero 90% of wing pouch). Use wing size to quanitfy amount of regeneration.
  • Tune wildtype response to have 50% regeneration
  • Find that Trithorax dominant increases regeneration and Trx mutant decreases.
  • trx mutants regenerate half as much as wildtypes.
  • trx hets still form regenerating blastemea — same amount of tissue is regenerating as in wildtype.
  • tissue is not dying
  • regenerating animals delay pupariation (depndent of insulin like peoptide 8 and retinoid signaling). Trx hets delay for less long.
  • rescue pupation delay using UAS dilp8 — regenerate more wing.
  • dlip8 is regulated by JNK signaling.

How is growth coordinated to maintain isometry (proper size and proportion)

  • growth rate of eye reduced when wing disk is damaged and has to regrow.
  • damaged imaginal tissues release dilp8 to coordinate growth.
  • NOS overexpression also reduces growth rate of disks.
  • experiment: irraiate larva, shield eyes with lead tape.
  • NOS is necessary for growth delay in eye (remove NOS lose delay)
  • NOS is not required for developmental delay but is a mediator of dilp8
  • propose NOS is inhibitory on growth during feeding and activating afterwards.
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Fly Meeting, Wed 3/4/15

Opening Session

(7:00 pm – 9:15 pm, 3/4/15)

Opening statements

  • Adam Fagen, Exec. director GS
  • 1600 registrants, 36% faculty, 16% postdoc
  • 30 countries, 46 states
  • Next Fly meeting is in July 2016. Joint sessions with other genetics (including Denis Duboule and Francis Collins)
  • Wangler … Bellen 2015 Fruit Flies in Biomedical Research: The value of Fly research

Fprmer Levine lab members here

  1. Angelike Stathopolous
  2. Jessica Cande
  3. Dave Hendrix
  4. Christine Rushlow
  5. John Reinitz
  6. David Arnosti
  7. Tony Ip
  8. Alistair Boettiger
  9. Scott Barolo
  10. Matthias Mannervik
  11. Michelle Markstein
  12. Haini Cai
  13. Albert Erives

Larry Sandler Award Speaker


  • Zhao Zhang
  • PI at Carnegie Institute (as Spradling)
  • Genome faces challenges from outside
  • High number of transposons.
  • piRNAs inhibit transpsons to maintain genome stability.
    • 23 – 30 nt.
    • associate with Piwi and Ago3
    • mainly function in germline
    • come from large pi RNA clusters (142 clusters in genome).
  • how to sort piRNA precursors into piRNAs and not translate.

Rhino Protein

  • mutant named as they make single dorsal appendage
  • rhi ChIP-seq (didn’t work for months)
  • Very strong correlation between rhi ChIP-seq and PiRNA production across the genome.
  • 131 of 142 clusters correlate reduction in piRNA in rhino mutant and amount of rhino binding (peak height)
  • Rhi binding surpresses splicing
  • spliced transcripts lose piRNA production
  • Rhino binding can convert coding gene Sox2F into a piRNA precursor.
  • mutate Rhino, all transcripts are spliced
  • Rhi LacI fusion represses gene expression
  • anti-sense transcription also necessary for piRNA generation?
  • cap-binding protein (required for splicing) recruited to 5′ end of protein coding genes
  • rhino associates 5′, blocks cap-binding protein (homologous but enzyamtically inactive).


  • Rhino has chromodomain and chromo-shadow, bot doesn’t bind heterochromatin. How is it targeted?
  • what happens if you tether HP1 to LacI? Also silencing?
  • Why need to block splicing for piRNA generation?
    • similar to other otrganisms use piRNAs

Key Note

  • Allan Spradling
  • BA in physics at Chicago
  • Went to Carnegie after his post doc and has been there since.
  • HHMI for 27 years
  • 168 publications. 1982 paper with Rubin: transformation based on transposable elements (pElement transgenesis)

Title: Drosophila: asuming the mantle of leadership in Biology

  • An era of unprecedented progress in genetics.
  • no cloned genes, no sequencing of DNA (let alone genomes)
  • expanded scope of and awareness of genetics

How did this growth happen?

  • Vannevar Bush and James Shannon
  • Even the 50s NIH had a multimillion dollar budget, most given to doctors. These guys decided to support Ph.D.s
  • NIGMS lead to an explosion of basic biology
  • Spradling, first grant at 30, (claims given for accomplishment not writing).

why don’t we feel better about the future?

  • gov is seriously considering a return to the earlier form of NIH
  • logical continuation: continue with model organisms
  • new human / mammalian-cell centric vision.

How much conservation really is there?

  • Mouse ovary (superficially) looks very different than fly ovary in structure and behavior
  • Drosophila: 16 cell cyst -> become 15 NCs and 1 OC.
  • motors fire up and a lot of things start moving from all those nurse cells into the oocyte (mitochondria, RNPs and others)
  • Ring canals in 16 cell mouse oocyte follow the same steps of transport. Single germ cell labeling and lineage tracing in the mouse.
  • Mouse cyst are unstable, don’t remain connected. Could make multiple follicle cells
  • find more cells get multiple centrosomes (get downstream stuff pumped into them).
  • Cells lacking Balbiani body golgi (follicle precursor) apoptose.
  • Mouse oocytes may start life unequal. (Early eggs come from big cysts and are of higher quality?)
  • No germline stem cells in mice.
  • despite ‘quack stem cell treatments’ that offer to proliferate stem cells in coordination with IVF.
  • 32 mature eggs on each ovary, only 1 can be released at a time. One is always protruding more and is uniquely expressing high levels of metalloprotease
  • Drosophila ‘corpus lutium’, steroid produced from remaining follicle cells. Only one remains at a time.
  • homolog of fallopian tube
  • oocytes like to load up on lipids. Leads to problems in humans generating infirtility
  • Fly produces proteins and substantial amount of lipid (stage 10) into oocyte. This requires substantial amount of the flies metabolic resources
  • controlled by an ectodysin pulse.
  • Corresponding pathway is used in mammalian liver. Is it also use in mammalian reproduction / oogenesis?
  • Drosophila females increase substantially in fat once the ovaries are sexually mature (as in humans).
  • this change is driven by the brain. presence of ovaries leads to higher ectodysin release.


  • Could never have anticipated this level of conservation. Built ever since the Hox genes — tissue and physiology conservation.
  • existence of this unexpected conservation is also pretty cool science.
  • every college graduate should be aware of this remarkable conservation.
  • Best thing to do is discover something new.

Mammals and polyploid cells

  • get new polyploid cells in heart attack.
  • wounded flies cells around the wound become polyploid — EdU replication up, but no cell division.
  • scales with wound size. replaces the number of genomes that were lost in the wound.
  • cells persist for life time. Process contributes to wound healing — healing is worse if you knock out this response.
  • Hippo pathway required
  • overexpresion of hippo get more than normal polyploidy.
  • polyploid cells in aging and wounding might be a general stress response?
  • hypothesize that this restores mechanical stability.
  • Polyploid cells different levels of genes due to under-replication. Proposed nested forks, factors of 2
  • salivary gland under-replication
  • arrested forks are not actually stable — cells repair these by break and religation. Test this by sequencing. Confirmed. creates deletions in low replicating regions. These sum deletions are what create the low read count of under-replication.
  • All these breaks create extensive genomic novelity. generates diversity in IgG regions and others (?)

Superior tools in flies

  • MIMICs make GFP lines.
  • swapped in Hsp70 promoter into lots of sites (heterochromatic and non heterochromatic) Under heat shock, some cell types turn on GFP and some don’t. chromatin state mapping in cell type specific way.
  • ‘not going to understand biology better until we get better understanding of gene regulation and genomics’


  • tissue culture cell models for multicellular interactions has not played through
  • tissue culture models wouldn’t need to be cut by a large fraction to increase model organism work by a large fraction.
  • should do more cross-system research
  • convincing society will require great leadership, but alternative will be disasterous.
  • billions of dollars spent on GWAS (mammalian centered technology). Follow which genes segregate and relate to populations. Should have talked to Drosophila pop bio first. 100 million dollar study in height explains less than 1% of variation with a BMP receptor link (could have guessed?)
  • lots of stuff maps to non-regulatory regions (which we don’t know very much about in human / mammals)
  • ENCODE – no biological questions.
  • human genetics wants to go from genome to disease. Need to look at development — tried this already in model organism, a useful model will need to look at the genes in their tissue context.
  • least favorite nucleomics. 3C, 5C ‘after all the aggregation artifacts’. Project is “limited to mammalian cells”.
    • come on, we’ve learned the best about this stuff from model organisms. If you are going to do this, at least do it in a model organism.


  • education about evolution. First, its surprising, don’t make people feel bad or negative. We just need to spread the idea better.
  • First mission statement of NIH is to understand what underlies the biology. No statement about health in that mission statement, only the second statement is health relevant.
  • we need to step up the efforts. Tell them more of the science.
  • Model organism people need to work together and talk together more. Our community is not so visible.


  • GSA medal: Heinikoff (last 15 years) and before.
  • Education award: (chair at U. Minnasota)
  • Thomas Hunt Morgan Career award: Brian Charlesworth — origin of sex chromosomes, sex chromosome evolution. advanced concept of genetic load of deleterious mutations. pioneer in studying aging and the evolution of aging.
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Monday 03/02/15

9:00 am – 12:30 am

Oligo Secondaries

Revising manuscript

  • More failures of microsoft word: MSO recommends I change my whole Windows Settings language for the bloody desktop just to get the MS word spell checker to work. Stupid program, I have multiple manuscripts some which need to be written in U.K. English and some which need to be in U.S. English and I don’t want to reboot my editor and change my windows configuration every time I want to switch between the too.
  • restarted revisions based on CW replies to XZ
  • finalized revisions, sent to CW

Revised Fig 2

  • changed panels for one with smaller FWHM
  • redid code to make things smoother
  • changed formatting to match Fig 3 a bit better

Ph Project

  • reviewed latest draft of manuscript.
  • Re-read all STORM main text and methods sections and all ChIP-seq 4C comparison main text and supplement / methods sections.
  • reviewed all figures
  • sent comments to NF.

Chromatin project

Meeting prep

  • discussed slides for both talks with Bogdan
  • discussed Hi-C data, reviewed new code.
  • working on slides!
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