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Lecture 8 • Lab results/lab report • The morphogen problem • Nuclear gradients and linear pathways • b-TGF and Brinker • Three ‘habits’ of signaling pathways Results are posted on the web Go to results page Click and find your files Instruction to authors Instruction to authors Summary /Abstrac t: This section cons tit utes a br ief description of the entire study, including the background or motivation, the methods, result s and conc lusions. It is often easier to complete the abstract after the rest of the paper is complete and all of the results are in place, but you shou ld still try to create a rough draft of the abstract at the beginning. When your abstract is finis hed, use the following check list to ensure that it is appropriate: Subject of the pap er is stated immediately Scope and ob jectives are identifi ed Significant find ings are summarized All abbrevia tions are defined No referenc es are cited No mention of figur es or tables from the main text I. INTRODUCTION Main po int of fir st paragraph : Wha t problem are you looking a t: the larger picture. Turnitin.com • To be marked the lab report must be submitted to turnitin.com minus the references. • Class ID: see web site • Enrollment password: see web site • Due April 5, 2012 The problem with Morphogens 100% Percent bound/ response 50% Concentration The problem with Morphogens 100% Percent bound/ response 50% Concentration The problem with Morphogens 100% Percent bound/ response 50% Concentration The problem with Morphogens Cooperative 100% Percent bound/ response 50% Concentration The problem with Morphogens Cooperative 100% Percent bound/ response 50% Concentration The primacy of secreted morphogens Teleman and Cohen Cell 103, 971 bTGF/DPP pathway bTGF/DPP Type1. thickvein ligand Type2, punt Smads One eyed pinhead oep Co-receptor in zebrafish The primacy of secreted morphogens Teleman and Cohen Cell 103, 971 Problems with primacy of secreted morphogens • Changes to gene expression is ultimately a nuclear event. • Most morphogen signaling pathways are linear. • Gradient on gradient on gradient. • Bicoid rules. Toll Dorsal Pathway Gilbert 7th Ed. Gradients • Spatzle is activated in a graded manner. • Toll is active in a graded manner. • Pelle kinase is active in a graded manner. • Cactus is degraded in a graded manner. • Dorsal enters the nucleus in a graded manner. Dorsal nuclear gradient Stathopoulos and Levine Dev. Biol. 246, 57 Interpretation in the nucleus of the gradient Stathopoulos and Levine Dev. Biol. 246, 57 Interpretation in the nucleus of the gradient Convert low affinity site to high affinity Stathopoulos and Levine Dev. Biol. 246, 57 Interpretation in the nucleus of the gradient Stathopoulos and Levine Dev. Biol. 246, 57 Dorsal patterns the dorsal ventral axis Stathopoulos and Levine Current opinion in Genetics and Development 14, 477 Different Dorsal dependent regulatory elements Stathopoulos and Levine Current opinion in Genetics and Development 14, 477 Different Dorsal dependent regulatory elements Stathopoulos and Levine Current opinion in Genetics and Development 14, 477 Problem with Dorsal as model morphogen • The Toll Dorsal pathway activated at syncytial blastoderm stage. • Look at DPP again. bTGF/DPP pathway bTGF/DPP Type1. thickvein ligand Type2, punt Smads 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 What do Mad and Medea do in the nucleus? • Activate expression of an inhibitory Smad called Dad • Repress Brinker expression Brinker? • Identified as a DPP regulated gene required for the repression of DPP regulated genes. Brinker expression repressed by DPP pathway FRT mad FRT UbiGFP Campbell and Tomlinson Cell 96, 553 Brinker represses Octomotor blind and Spalt FRT brkXH FRT UbiGFP Campbell and Tomlinson Cell 96, 553 Brinker epistasis Jazwinska et al., Cell 96, 563 Brinker is a nuclear protein that is repressed by DPP such that it is expressed in a DPP antigradient. Sal and omb are repressed by different concentrations of Brinker. Campbell and Tomlinson Cell 96, 553 Brinker DNA binding domain bound to DNA Questions about Brinker • How do Mad and Medea both activate and repress transcription? • How does the DPP pathway regulate Brinker expression to create the antigradient? Schnurri is required for expression of DPP responsive genes Schnurri phenotype is suppressed by brinker mutant wt shn brk shn, brk Marty et al., Nature cell biol. 2, 745 Expression in shn brk mutants GFP shn Sal expression shn Brinker expression shn brk Sal expression Marty et al., Nature cell biol. 2, 745 DPP pathway has two branches Marty et al., Nature cell biol. 2, 745 Looking for the brinker regulatory element Muller et al., Cell 113, 221 24 bases required for repression Pyrowolakis et al., Dev. Cell 7, 229 Mad Medea and Schnurri bind to the silencer Pyrowolakis et al., Dev. Cell 7, 229 Brinker is not the only gene repressed Pyrowolakis et al., Dev. Cell 7, 229 Brinker is a nuclear protein that is repressed by DPP such that it is expressed in a DPP antigradient. Sal and omb are repressed by different concentrations of Brinker. Bicoid rules Campbell and Tomlinson Cell 96, 553 The three ‘habits’ • Barolo and Posakony 2002 • Looking for common themes in the organization of signaling pathways. Trying to explain the precision of expression Example wingless expression The three ‘habits’ • Activator insufficiency • Cooperative activation • Default repression SPRE: Signaling pathway response elements SPRE-binding transcription factor SPRE: Signaling pathway response elements SPRE-binding transcription factor HH: Ci/Gli WNT: Lef/Tcf Notch: Su(H)/CBF1 Other factors Barolo and Posakony Genes and Dev. 16, 1167 3 habits model Barolo and Posakony Genes and Dev. 16, 1167 Basis for the proposal of the model Barolo and Posakony Genes and Dev. 16, 1167 Activator insufficiency • SPRE-binding transcription factor can not activate transcription alone. • Tissue culture vs in vivo Cooperative activation • SPRE-binding transcription factors require other transcription factors for the activation of transcription. • The interaction is cooperative Default repression • In the absence of ligand SPRE-binding factors repress transcription. 3 habits model Barolo and Posakony Genes and Dev. 16, 1167 Switching from default repression to activation Barolo and Posakony Genes and Dev. 16, 1167 3 habits model Barolo and Posakony Genes and Dev. 16, 1167 Consequences of the 3 ‘habits’ Barolo and Posakony Genes and Dev. 16, 1167 Trying to explain the precision of expression Wingless is regulated by the HH pathway DPP pathway and the three habits Pyrowolakis et al., Dev. Cell 7, 229