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M267 February 2008 Larry Zipursky Lecture 5 Patterning Tissue through

M267 February 2008 Larry Zipursky Lecture 5 Patterning Tissue through Graded Extracellular Signals. Very important message!. Instead of tomorrow’s lecture there will be a 2 hr review session in BSRB 154 from 10 - 12 am. This will be in a “question and answer” format on lectures 1-5.

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M267 February 2008 Larry Zipursky Lecture 5 Patterning Tissue through

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  1. M267 February 2008 Larry Zipursky Lecture 5 Patterning Tissue through Graded Extracellular Signals

  2. Very important message! Instead of tomorrow’s lecture there will be a 2 hr review session in BSRB 154 from 10 - 12 am. This will be in a “question and answer” format on lectures 1-5.

  3. Dissecting the Mechanism of Dpp-mediated Brinker Repression Fusion of regulatory regions to LacZ brinker gene Upstream regulatory region coding The “C” fragment can repress translation of other promoters in an orientation and gene independent fashion in response to Dpp. Muller et al. (2003). Cell 113-221-233

  4. Repressing brinker requires mad, med, tkv and schnurri FRT-mediated recombination to make schnurri mutant patches of cells (Mad clones made in a similar way). FRT 42 schnurri FRT 42 GFP Heat shock Induction of Flp Green cell: GFP Red: (schnurri genotype) Green cell: schnurri + Not Green: schnurri - Lac Z dpp enhancer brk silencer (the “C” fragment) The dpp enhancer is a variant that is expressed ubiquitously in the wing pouch. Muller et al. (2003)

  5. Mad, Medea and Schnurriform a Complex on the BrinkerRepressor Sequences Gel Shift Assay Co-transfection of Drosophila S2 cells with activated Dpp receptor (TkvQ235D) and plasmids encoding different proteins. Test extracts for binding to 32P-labeled brinker repressor sequences. S2 cell extracts Muller et al. (2003)

  6. Transcriptional Readouts in Response to the Dpp Gradient: Activation results from Repression of Repression Protein distribution determined by antibody staining Differential response of omb and sal reflect differences in number and strength of repressor and activator sequences Affolter and Basler (2007)

  7. Summary 1. Induction is graded not sequential. 2. Graded signaling leads to graded nuclear entry of a nuclear repressor complex. 3. Dpp activates a repressor of a repressor.

  8. Hedgehog Signaling Varjasalo and Taipale (2007)

  9. Hedgehog is Cleaved Lee et al (1994)

  10. Cleavage is Necessary for Hh Activity: Dual Function? Dpp marker in blue In Porter et al (1995): Ectopic expression of N - induction Ectopic expression of C - no induction WT Hh Heat shock Hh What tethers Hh to the membrane? Heat shock HhH329A (Lee et al. 1994) (Not cleaved)

  11. Cholesterol is the Nucleophile in vivo: Membrane tethering of Hh Cleavage Pathway Assay for Lipid Attachment In the presence of 50 mm DTT cleavage is independent of lipid In the presence of 1 mM DTT cleavage is dependent upon lipid Porter et al (1996)

  12. dispatched (disp) is Required in Posterior Clones to Pattern Anterior Regions Generation of clones: f hs-flp; FRT disp/FRT M P[f] f=forked mutant; M=minute (causes growth disadvantage to heterozygous tissue and cell lethal when homozygous) P[f] = wild type forked transgene Burke et al. 1999

  13. disp, like hh, is required in posterior cells Genetic mosaics in discs: Wild type tissue is green. Mutant tissue is NOT green. Burke et al. 1999

  14. disp is required for release but not proteolytic cleavage of Hh Larval genotype WT hhFA in WT hhFA in disp Hh-NHA in WT Apical Sections through wing disc Basal Apical Burke et al. 1999 A Basal

  15. Removal of cholesterol circumvents disp requirement for Hh release Hh-NHA expressed in the posterior compartment drives Dpp-lac Z expression throughout anterior compartment Hh-NHA is bound by patched Burke et al. 1999 Expanded anterior compartment without cholesterol linkage

  16. disp acts specifically on cholesterol tethered Hh Testing a non-lipid attachment to the membrane en-Gal4 driving expression of UAS hh-CD2 Testing another lipid attachment to the membrane Actin 5C>CD2>Gal4 driving UAS transgenes with membrane tethered and free Hh. Burke et al. 1999

  17. Cholesterol-modification of Hh is Critical for Correct Patterning of both High and Low Threshold Targets Cholesterol No cholesterol (cholesterol) (no cholesterol) Method Actin 5C>CD2>Gal4 UAS-Hh, UAS-gal Callejo et al (2006)

  18. High Threshold Targets Require HSPG High threshold targets not induced in clones Low threshold targets induced in clones MARCM: Green lack HSPG and express lipid modified Hh Callejo et al. (2006)

  19. Distribution of Hh is Modulated by Cholesterol and HSPGs Interaction between cholesterol modified Hh and HSPGs is crucial for patterning gradient Fully processed Hh Cleaved Hh without lipid attachment Guerrero and Chiang, 2006

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