Lab results/lab report

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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

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Results are posted on the web Go to results page
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Click and find your files
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Instruction to authors
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Instruction to authors
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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

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The problem with Morphogens
100
Percent bound/ response
50
Concentration
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The problem with Morphogens
100
Percent bound/ response
50
Concentration
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The problem with Morphogens
100
Percent bound/ response
50
Concentration
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The problem with Morphogens
Cooperative
100
Percent bound/ response
50
Concentration
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The problem with Morphogens
Cooperative
100
Percent bound/ response
50
Concentration
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The primacy of secreted morphogens
Teleman and Cohen Cell 103, 971
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bTGF/DPP pathway
bTGF/DPP
Type1. thickvein
ligand
Type2, punt
Smads
One eyed pinhead oep Co-receptor in zebrafish
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The primacy of secreted morphogens
Teleman and Cohen Cell 103, 971
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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.

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Toll Dorsal Pathway
Gilbert 7th Ed.
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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.

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Dorsal nuclear gradient
Stathopoulos and Levine Dev. Biol. 246, 57
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Interpretation in the nucleus of the gradient
Stathopoulos and Levine Dev. Biol. 246, 57
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Interpretation in the nucleus of the gradient
Convert low affinity site to high affinity
Stathopoulos and Levine Dev. Biol. 246, 57
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Interpretation in the nucleus of the gradient
Stathopoulos and Levine Dev. Biol. 246, 57
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Dorsal patterns the dorsal ventral axis
Stathopoulos and Levine Current opinion in
Genetics and Development 14, 477
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Different Dorsal dependent regulatory elements
Stathopoulos and Levine Current opinion in
Genetics and Development 14, 477
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Different Dorsal dependent regulatory elements
Stathopoulos and Levine Current opinion in
Genetics and Development 14, 477
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Problem with Dorsal as model morphogen

• The Toll Dorsal pathway activated at syncytial
  blastoderm stage.
• Look at DPP again.

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bTGF/DPP pathway
bTGF/DPP
Type1. thickvein
ligand
Type2, punt
Smads
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bTGF/DPP pathway
bTGF/DPP
P
Type1. thickvein
ligand
Type2, punt
Smads
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bTGF/DPP pathway
bTGF/DPP
P
Type1. thickvein
ligand
Type2, punt
P
Smads
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bTGF/DPP pathway
bTGF/DPP
P
Type1. thickvein
ligand
Type2, punt
P
Smads
Mad Medea
Nucleus
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What do Mad and Medea do in the nucleus?

• Activate expression of an inhibitory Smad called
  Dad
• Repress Brinker expression

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Brinker?

• Identified as a DPP regulated gene required for
  the repression of DPP regulated genes.

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Brinker expression repressed by DPP pathway
FRT
mad
FRT
UbiGFP
Campbell and Tomlinson Cell 96, 553
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Brinker represses Octomotor blind and Spalt
FRT
brkXH
FRT
UbiGFP
Campbell and Tomlinson Cell 96, 553
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Brinker epistasis
Jazwinska et al., Cell 96, 563
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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
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Brinker DNA binding domain bound to DNA
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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?

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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
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Expression in shn brk mutants
GFP
shn
Sal expression
shn
Brinker expression
shn brk
Sal expression
Marty et al., Nature cell biol. 2, 745
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DPP pathway has two branches
Marty et al., Nature cell biol. 2, 745
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Looking for the brinker regulatory element
Muller et al., Cell 113, 221
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24 bases required for repression
Pyrowolakis et al., Dev. Cell 7, 229
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Mad Medea and Schnurri bind to the silencer
Pyrowolakis et al., Dev. Cell 7, 229
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Brinker is not the only gene repressed
Pyrowolakis et al., Dev. Cell 7, 229
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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
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The three habits

• Barolo and Posakony 2002
• Looking for common themes in the organization of
  signaling pathways.

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Trying to explain the precision of expression
Example wingless expression
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The three habits

• Activator insufficiency
• Cooperative activation
• Default repression

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SPRE Signaling pathway response elements
SPRE-binding transcription factor
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SPRE Signaling pathway response elements
SPRE-binding transcription factor
HH Ci/Gli WNT Lef/Tcf Notch Su(H)/CBF1
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Other factors
Barolo and Posakony Genes and Dev. 16, 1167
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3 habits model
Barolo and Posakony Genes and Dev. 16, 1167
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Basis for the proposal of the model
Barolo and Posakony Genes and Dev. 16, 1167
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Activator insufficiency

• SPRE-binding transcription factor can not
  activate transcription alone.
• Tissue culture vs in vivo

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Cooperative activation

• SPRE-binding transcription factors require other
  transcription factors for the activation of
  transcription.
• The interaction is cooperative

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Default repression

• In the absence of ligand SPRE-binding factors
  repress transcription.

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3 habits model
Barolo and Posakony Genes and Dev. 16, 1167
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Switching from default repression to activation
Barolo and Posakony Genes and Dev. 16, 1167
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3 habits model
Barolo and Posakony Genes and Dev. 16, 1167
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Consequences of the 3 habits
Barolo and Posakony Genes and Dev. 16, 1167
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Trying to explain the precision of expression
Wingless is regulated by the HH pathway
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DPP pathway and the three habits
Pyrowolakis et al., Dev. Cell 7, 229