Fly Meeting, Sunday 3/8/15

Chris Rushlow

Intro

• 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?