lecture 8 lab results/lab report the morphogen problem nuclear gradients and linear pathways tgf...
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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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Instruction to authors
Instruction to authorsSummary/Abstract:
This section constitutes a brief description of the entire study, including the background
or motivation, the methods, results and conclusions. 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
should still try to create a rough draft of the abstract at the beginning. When your
abstract is finished, use the following checklist to ensure that it is appropriate:
Subject of the paper is stated immediately
Scope and objectives are identified
Significant findings are summarized
All abbreviations are defined
No references are cited
No mention of figures or tables from the main text
I. INTRODUCTION
Main point of first paragraph: What problem are you looking at: the larger picture.
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The problem with Morphogens
100%
50%
Percentbound/response
Concentration
The problem with Morphogens
100%
50%
Percentbound/response
Concentration
The problem with Morphogens
100%
50%
Percentbound/response
Concentration
The problem with Morphogens
100%
50%
Percentbound/response
Concentration
Cooperative
The problem with Morphogens
100%
50%
Percentbound/response
Concentration
Cooperative
The primacy of secreted morphogens
Teleman and Cohen Cell 103, 971
TGF/DPP pathway
TGF/DPP
ligand
One eyed pinhead oepCo-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
MadMedea
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
FRT UbiGFP
mad
Campbell and Tomlinson Cell 96, 553
FRT
FRT UbiGFP
brkXH
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
wt shn brk shn, 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/GliWNT: Lef/TcfNotch: 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