Lecture 8 Lab results/lab report The morphogen problem Nuclear gradients and linear pathways  TGF and Brinker Three ‘habits’ of signaling pathways

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The problem with Morphogens 100% 50% Percent bound/ response Concentration

The problem with Morphogens 100% 50% Percent bound/ response Concentration

The problem with Morphogens 100% 50% Percent bound/ response Concentration

The problem with Morphogens 100% 50% Percent bound/ response Concentration Cooperative

The problem with Morphogens 100% 50% Percent bound/ response Concentration Cooperative

The primacy of secreted morphogens Teleman and Cohen Cell 103, 971

 TGF/DPP pathway  TGF/DPP ligand One eyed pinhead oep Co-receptor in zebrafish Type1. thickvein Type2, punt Smads

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.

 TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads

 TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads P

 TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads P P

 TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads P P Nucleus Mad Medea

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 UbiGFP mad Campbell and Tomlinson Cell 96, 553

FRT UbiGFP brk XH Brinker represses Octomotor blind and Spalt 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 anti- gradient. 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 anti- gradient?

Schnurri is required for expression of DPP responsive genes Schnurri phenotype is suppressed by brinker mutant wtshnbrkshn, brk Marty et al., Nature cell biol. 2, 745

Expression in shn brk mutants shn Sal expression shn Brinker expression shn brk Sal expression GFP 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 anti- gradient. 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-binding transcription factor SPRE: Signaling pathway response elements

SPRE-binding transcription factor SPRE: Signaling pathway response elements 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