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Lecture 7 Advanced epistasis Ectopic expression in Drosophila Morphogens: secreted ligands
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Epistasis headlesstwinhead
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Epistasis headless hltwinhead th hl lf th lf phenotype no head; two tails no tail; two heads
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Epistasis headless hltwinhead th hl lf th lf phenotype no head; two tails no tail; two heads Head Tail
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Advanced Epistasis Why do I always assume that the most epistatic step is at the end of the pathway?
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Advanced Epistasis Why do I always assume that the most epistatic step is at the end of the pathway? Can we always assume this?
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Example biochemical pathway ABCDE WXY Z E is an essential metabolite
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Phenotype of mutants in W, X, Y, Z all can not grow without E in the media.
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Phenotype of mutants in W, X, Y, Z all can not grow without E in the media.
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Phenotype of double mutants in W, X, Y, Z.
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Order of epistasis Z epi to Y epi to X epi to W
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Example biochemical pathway ABCDE WXY Z What intermediates accumulates in the mutant?
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Accumulated intermediates
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Double mutants
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Order of epistasis W epi to X epi to Y epi to Z
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Order of epistasis W epi to X epi to Y epi to Z Z epi to Y epi to X epi to W
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Order of epistasis W epi to X epi to Y epi to Z Z epi to Y epi to X epi to W When you assay along the pathway When you assay at the end of the pathway
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Ectopic expression in Drosophila Heat-shock promoter fusion genes Binary or two component system Flip-mediated ectopic expression Combination system I Combination system II
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Heat-shock promoter fusion gene YFG hsp Qualities A) high level of expression B) expressed in all cells C) expression is transient
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Binary or two component system Fly stock A: UAS lineFly stock B: Driver line YFG GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer YFG GAL4 Progeny
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Binary or two component system Fly stock A: UAS lineFly stock B: Driver line YFG GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer YFG GAL4 Progeny GAL4
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How do you make different GAL4 drivers? A. Construct them Known promoter GAL4
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How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. lacZ p P-element transposase promoter: very very weak expression-basically no expression by itself. Enhancer detector
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Enhancer structure of a portion of the genome Gut Brain Gonad
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Insertion of a enhancer detector lacZ Gonad Gut Brain
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Insertion of a enhancer detector Gonad Gut Brain lacZ
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How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. GAL4 p P-element transposase promoter: very very weak expression-basically no expression by itself. Enhancer detector
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Qualities of Binary system Control temporal and spatial pattern of YFG Expression is not transient Nonexpression of YFG from UASGAL4 allows transformants to be established
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YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression FLP hsp Heat shock and induce FLP expression
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YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression
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YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression
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YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression Results in a mosaic pattern of expression of YFG: recombination does not occur in all cells
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Combination system I YFG FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 Promoter or enhancer
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Combination system I YFG FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 Promoter or enhancer Induce FLP expression
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Combination system I YFG FRT UASGAL4 GAL4 Promoter or enhancer
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What happens? Cell expressing GAL4Cells that have undergone a site specific recombination event
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What happens?
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Combination system II GAL4 FRT y+y+, CD2 Tubulin promoter YFG UASGAL4 FLP hsp
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Combination system II GAL4 FRT y+y+, CD2 Tubulin promoter YFG UASGAL4 FLP hsp Induce FLP expression
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Combination system II GAL4 FRT Tubulin promoter YFG UASGAL4
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Combination system II GAL4 FRT Tubulin promoter YFG UASGAL4 Results in clones of cells that express YFG
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Morphogen Alan Turing-a father of computational sciences In 1952 publishes a paper entitled “A. M. Turing on the chemical basis of morphogenesis.” Not recommended reading
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[morphogen] Simplified Turing morphogen model
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[morphogen] Simplified Turing morphogen model Fate A Fate B
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Morphogen Long range action Forms a concentration gradient of activity Affect polarity Concentration dependent response Different fates at different concentrations
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Suspected secreted morphogens Tumor growth factor TGF/decapentaplegic dpp WNT/wingless wg Sonic hedgehog etc./hedgehog hh
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TGF/DPP pathway TGF/DPP ligand One eyed pinhead oep Co-receptor in zebrafish Type1. thickvein Type2, punt Smads
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One eyed pinhead Schier et al. 1997 Development
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Get similar phenotypes in mice by altering BMP signalling Gilbert Developmental Biology 7th ed
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TGF/DPP pathway TGF/DPP ligand Type1. thickvein Type2, punt Smads P
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TGF/DPP pathway TGF/DPP ligand Type1. thickvein Type2, punt Smads P P
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TGF/DPP pathway TGF/DPP ligand Type1. thickvein Type2, punt Smads P P Nucleus Mad Medea
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DPP a morphogen? Long range action YES Forms a concentration gradient of activity YES Affect polarity YES Concentration dependent response YES Different fates at different concentrations YES
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Wing imaginal discs
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A P
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DPP is expressed in gradient Wing imaginal disc dpp mRNA DPP protein
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Spalt is a DPP responsive gene Increase [DPP] and spalt response widens
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Spalt and octomotor blind are DPP responsive SPT OMB
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Spalt and octomotor blind are DPP responsive SPT OMB SPT
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Long range action DPP expressed in this cell
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Long range action DPP expressed in this cell Directly Affects expression in this cell
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Signal relay system DPP expressed in this cell indirectly
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TGF/DPP pathway TGF/DPP ligand One eyed pinhead oep Co-receptor Type1. thickvein Type2, punt Smads
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Combination system I tkv Q253D FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 ubiquitous expression
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UAS-tkv gf CD2 expressionOMB expression
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UAS-tkv gf CD2 expressionOMB expression
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FLP mediated mitotic recombination with a tkv lf allele
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TKV is required right out to the edge of OMB expression domain
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Signal relay system DPP expressed in this cell indirectly
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no tail expression squint sqt Zebrafish example
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no tail expression squint sqt Zebrafish example one eyed pinhead mutant
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no tail expression squint sqt Zebrafish example one eyed pinhead mutant Wild-type cell
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no tail expression squint sqt Zebrafish example one eyed pinhead mutant Wild-type cell
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