L4 - Morphogens

Question 1

What is a morphogen?

Answer 1

A soluble secreted molecule that acts at a distance to specify the fates of cells
May specify more than one cell type by forming a concentration gradient
Increasing/decreasing morphogen concentration continually alters the pattern

Question 2

What led to the discovery of morphogen gradients?

Answer 2

In the molecular age, biologists cloned ligands that they thought might act as morphogens
E.g. a gene that when mutated results in loss of patterning

Question 3

What are the two requirements of a morphogen?

Answer 3

Induce different outputs at different concentrations

Act directly at a distance

Question 4

Requirements of a morphogen - induce different outputs at different concentrations

Answer 4

Instructive signal
Permissive signal – not morphogens
- Cell already knew its fate just needs a signal to tell it to assume this fate
- Red signal permits cells to respond to previously inherited cell fate determinant

Question 5

Requirements of a morphogen - act directly at a distance

Answer 5

Red ligand from red cell
- One signal does everything
Bucket brigade – not used by morphogens
- Each signal induces another

Question 6

What are the two ways to distinguish between instructive and permissive signals

Answer 6

Provide a second source of red signal

Provide red signal at a uniform concentration

Question 7

What happens if you provide a second source of red signal?

Answer 7

Instructive signal – get a mirror image patterned effect
Permissive signal – no effect
Experiments often done in chick wings – get a mirror image

Question 8

What happens if you provide a red signal at a uniform concentration?

Answer 8

Instructive signal – one cell fate induced

Permissive signal – no effect

Question 9

What are the two ways to distinguish between one signal and bucket brigade?

Answer 9

Genetically engineer proposed morphogen into a juxtacrine

Make a genetic mosaic that lacks the receptor for the red signal in one of the cells

Question 10

What happens if you genetically engineer proposed morphogen into a juxtacrine?

Answer 10

Add a transmembrane domain – signal cannot diffuse
One signal – only neighbouring cell receives signal
Bucket brigade – no effect

Question 11

What happens if you make a genetic mosaic that lacks the receptor for the red signal?

Answer 11

One signal – cell without receptor does not receive signal and does not differentiate
Bucket brigade – no effect (unless green cell lacks receptor)

Question 12

What establishes a shallow morphogen gradient?

Answer 12

Passive diffusion

Question 13

What establishes a steep morphogen gradient?

Answer 13

Binding to molecules in the ECM (heparan sulphate proteoglycans) and high concentrations of receptor - restricted diffusion
Rapid degradation of signal in the ECM

Question 14

Where are heparan sulphate proteoglycans found?

Answer 14

Found in the ECM and bind to many ligands

Question 15

How do HSPGs regulate morphogen diffusion?

Answer 15

Sequestration or slowing diffusion – e.g. BMP - TGFbeta

Facilitating diffusion - Hh

Question 16

What does planar transcytosis have a role in?

Answer 16

Role in establishing some morphogen gradients

Question 17

Planar transcytosis method

Answer 17

Pit forms in cell membrane and engulfs the morphogen in a vesicle
Repeated cycles of endocytosis and resecretion allows certain morphogens to travel through cells

Question 18

Evidence for transcytosis in Dpp (TGFbeta) signaling

Answer 18

Antibody staining shows that Dpp is found in vesicles

Mutations that block vesicle formation cause Dpp to act in a juxtacrine manner

Question 19

What is the role of timing in establishing morphogen gradients?

Answer 19

As the gradient is established, gene expression is changing

• Must be a mechanism to block premature specification
• Cell waits for the steady state of receptor activation to be achieved - molecular mechanism is poorly understood

Question 20

What is the transcriptional read out model used for?

Answer 20

Used to model how cells read or interpret a gradient to make a cell fate decision

Question 21

Transcriptional read out model

Answer 21

Higher concentration of morphogen = higher concentration of activated transcription factor
Receptor activation causes TF to enter the nucleus and direct transcription
- The same TF in every cell
- Concentration of 3 TF per nucleus results in green cell fate decision

Question 22

What is bicoid?

Answer 22

Is a morphogen and transcription factor

Question 23

Where is bicoid mRNA localised?

Answer 23

mRNA is localised at the anterior of the egg and is translated into protein during embryogenesis

Question 24

What happens to bicoid protein?

Answer 24

Diffuses through the cytoplasm and accumulates in nuclei of the syncytial blastoderm -generates a concentration gradient

Question 25

How is transcription factor concentration interpreted at the DNA level?

Answer 25

Enhancers that regulate green cell fate genes have lower affinity for TFs than those of blue cell fate genes

Question 26

Transcriptional activation in blue cell

Answer 26

Both sets of genes see the same medium levels of TF
However only high affinity enhancers can bind enough TF to activate gene expression
Blue cell fate genes are activated - high affinity
Green cell fate genes are inactive - low affinity

Question 27

Transcriptional activation in green cell

Answer 27

Green cell has high levels of TF in the nucleus

Both high affinity (blue) and low affinity (green) enhancers are activated

Question 28

Why doesn’t the green cell have blue characteristics?

Answer 28

Block blue gene expression by having one of the green cell fate genes encode a repressor – crosstalk regulation

Question 29

How are strict thresholds achieved when the gradient is not steep?

Answer 29

One of the blue genes encodes a transcription factor that activates its own expression – positive feedback regulation