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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Instruction to authors
Instruction to authors
S ummary/Abstract:
This sect ion const itutes a br ief descript ion of the ent ire study, including the background
or mot ivat ion, the methods, results and conclusions. It is often easier to complete the
abstract after the rest of the paper is complete and all of t he result s are in place, but you
should st ill t ry 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 ident ified

Significant findings are summarized

All abbreviations are defined

No references are cited

No mention of figures or tables from the main t ext
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%
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