Lecture 8 Lab results/lab report The morphogen problem Nuclear gradients and linear pathways TGF...

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 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