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Transcript
Lecture 7 • Advanced epistasis • Ectopic expression in Drosophila • Morphogens: secreted ligands Epistasis headless twinhead Epistasis headless hl twinhead th phenotype hl lf no head; two tails th lf no tail; two heads Epistasis headless hl twinhead th phenotype Head Tail hl lf no head; two tails th lf no tail; two heads Advanced Epistasis Why do I always assume that the most epistatic step is at the end of the pathway? Advanced Epistasis Why do I always assume that the most epistatic step is at the end of the pathway? Can we always assume this? Example biochemical pathway W A X B Y C Z D E is an essential metabolite E Phenotype of mutants in W, X, Y, Z all can not grow without E in the media. Phenotype of mutants in W, X, Y, Z all can not grow without E in the media. Mutant W - X - Y - - Z Added A to B Media C - - - - + - - - + + - - D + + + - E + + + + Phenotype of double mutants in W, X, Y, Z. Double mutants - - - ZW ZX - - - - - - ZY YW YX Added A to B Media C - - - - - - - - - - - - - - - D - - - + + E + + + + + - Order of epistasis Z epi to Y epi to X epi to W Example biochemical pathway W A X B Y C Z D E What intermediates accumulates in the mutant? Accumulated intermediates mutant - intermediate W A X- B - Y C Z- D Double mutants Double mutants - WX - WY - WZ - XY X-Z- YZ intermediate A A A B B C Order of epistasis W epi to X epi to Y epi to Z Order of epistasis W epi to X epi to Y epi to Z Z epi to Y epi to X epi to W Order of epistasis When you assay along the pathway W epi to X epi to Y epi to Z When you assay at the end of the pathway Z epi to Y epi to X epi to W Ectopic expression in Drosophila • Heat-shock promoter fusion genes • Binary or two component system • Flip-mediated ectopic expression • Combination system I • Combination system II Heat-shock promoter fusion gene hsp YFG Qualities A) high level of expression B) expressed in all cells C) expression is transient Binary or two component system Fly stock A: UAS line Fly stock B: Driver line Promoter YFG GAL4 or Enhancer GAL4 binding sites UASGAL4 GAL4 YFG Progeny Binary or two component system Fly stock A: UAS line Fly stock B: Driver line Promoter YFG GAL4 or Enhancer GAL4 binding sites UASGAL4 GAL4 GAL4 YFG Progeny How do you make different GAL4 drivers? A. Construct them Known promoter GAL4 How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. Enhancer detector p lacZ P-element transposase promoter: very very weak expression-basically no expression by itself. Enhancer structure of a portion of the genome Gonad Gut Brain Insertion of a enhancer detector Gonad Gut lacZ Brain Insertion of a enhancer detector Gonad Gut lacZ Brain How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. Enhancer detector p GAL4 P-element transposase promoter: very very weak expression-basically no expression by itself. 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 Flip mediated ectopic expression Tubulin promoter y+, CD2 FRT YFG FRT hsp FLP Heat shock and induce FLP expression Flip mediated ectopic expression Heat shock and induce FLP expression y+, CD2 FRT Tubulin promoter FRT YFG Flip mediated ectopic expression Heat shock and induce FLP expression y+, CD2 FRT Tubulin promoter FRT YFG Flip mediated ectopic expression Heat shock and induce FLP expression y+, CD2 FRT Tubulin promoter YFG FRT Results in a mosaic pattern of expression of YFG: recombination does not occur in all cells Combination system I UASGAL4 y+, CD2 FRT Promoter GAL4 or enhancer YFG FRT hsp FLP Combination system I UASGAL4 y+, CD2 FRT Promoter GAL4 or enhancer YFG FRT hsp FLP Induce FLP expression Combination system I UASGAL4 YFG FRT Promoter GAL4 or enhancer What happens? Cell expressing GAL4 Cells that have undergone a site specific recombination event What happens? Combination system II Tubulin promoter hsp y+, CD2 GAL4 FRT FRT UASGAL4 FLP YFG Combination system II Induce FLP expression hsp FLP Tubulin promoter y+, CD2 GAL4 FRT FRT UASGAL4 YFG Combination system II Tubulin GAL4 promoter FRT UASGAL4 YFG Combination system II Tubulin GAL4 promoter FRT UASGAL4 YFG Results in clones of cells that express YFG 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 Simplified Turing morphogen model [morphogen] Simplified Turing morphogen model [morphogen] Fate A Fate B Fate B Morphogen • Long range action • Forms a concentration gradient of activity • Affect polarity • Concentration dependent response • Different fates at different concentrations Suspected secreted morphogens bTumor growth factor TGF/decapentaplegic dpp WNT/wingless wg Sonic hedgehog etc./hedgehog hh bTGF/DPP pathway bTGF/DPP Type1. thickvein ligand Type2, punt Smads One eyed pinhead oep Co-receptor in zebrafish One eyed pinhead Schier et al. 1997 Development Get similar phenotypes in mice by altering BMP signalling Gilbert Developmental Biology 7th ed bTGF/DPP pathway bTGF/DPP P ligand Type1. thickvein Type2, punt Smads bTGF/DPP pathway bTGF/DPP P ligand Type1. thickvein Type2, punt P Smads bTGF/DPP pathway bTGF/DPP P ligand Type1. thickvein Type2, punt P Smads Mad Medea Nucleus 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 Wing imaginal discs Wing imaginal discs A P DPP is expressed in gradient dpp mRNA Wing imaginal disc DPP protein Spalt is a DPP responsive gene Increase [DPP] and spalt response widens Spalt and octomotor blind are DPP responsive SPT OMB Spalt and octomotor blind are DPP responsive SPT OMB SPT OMB Long range action DPP expressed in this cell Long range action Directly DPP expressed in this cell Affects expression in this cell Signal relay system indirectly DPP expressed in this cell bTGF/DPP pathway bTGF/DPP Type1. thickvein ligand Type2, punt Smads One eyed pinhead oep Co-receptor Combination system I UASGAL4 y+, CD2 FRT ubiquitous GAL4 expression tkvQ253D FRT hsp FLP CD2 expression UAS-tkvgf OMB expression CD2 expression UAS-tkvgf OMB expression FLP mediated mitotic recombination with a tkvlf allele FLP mediated mitotic recombination with a tkvlf allele TKV is required right out to the edge of OMB expression domain Signal relay system indirectly DPP expressed in this cell Zebrafish example squint sqt no tail expression Zebrafish example squint sqt one eyed pinhead mutant no tail expression Zebrafish example one eyed pinhead mutant Wild-type squint sqt cell no tail expression Zebrafish example one eyed pinhead mutant Wild-type squint sqt cell no tail expression
