Lecture 4- Morphogens

Question 1

What is a morphogen?

Answer 1

A soluble secrete molecule that acts at a distance to specify the fates of cells. A morphogen may specify more than one type by forming a concentration gradient

Question 2

What model do morphogens follow?

Answer 2

1. Source: cell initiates and maintains production of morphogen
2. Signal concentration gradient: morphogen diffuses away from the source down a concentration gradient
3. Sink: cells furthest from the source who receive the morphogen at the lowest concentration

Question 3

How is a cells fate determined by a morphogen threshold?

Answer 3

• Cells fate is determined by a threshold

* If the morphogen reaches the cell above a certain threshold, a certain fate is specified

Question 4

What happened when biologist cloned ligands they thought might act as a morphogen?

Answer 4

• A gene mutated resulted in the loss of patterning

* But not all molecules involved with patterning are morphogens

Question 5

Explain the pattern of cell fates with normal amount of morphogen

Answer 5

• As the amount of morphogen decreases, a new fate is specified
• The closer you are to the source/the higher the morphogen concentration, the more rapidly the cell fates will change with movement away from the source
• The further from the source/the lower the morphogen concentration, the cell fates will change less rapidly with movement away from the source
• The information is more dense by the source where the gradient is steeper

Question 6

Explain the pattern of cell fates with increased levels of morphogens

Answer 6

• The diffusion away from the source results in all the same cell fate as they are all above the highest threshold
• So will gain more cells where their fate is dependant on higher levels of morphogen
• A similar pattern is then followed as under ‘normal’ morphogen levels

Question 7

Explain the pattern of cell fates with reduced levels of morphogen

Answer 7

• Even at the highest level of reduced morphogen won’t be enough to specify certain cell fates
• Cell fates specified at higher concentrations/thresholds will be lost
• But there will be an increase in cells specified at low concentrations/thresholds

Question 8

Explain the pattern of cell fates with a long ranged morphogen

Answer 8

• Instead of decaying quickly, the morphogen diffuses further (e.g more stable protein)
• The leads to a flatter concentration gradient
• Less cell fates specified overall as the gradient can only specify certain cell fates within a smaller range

Question 9

Explain the pattern of cell fates where the morphogen is expressed ectopically

Answer 9

• Curved gradient

* Morphogen gradient moves away from the source but increases again as it moves closer to the ectopic source

Question 10

Explain the pattern of cell fates where the morphogen is mutated?

Answer 10

• No morphogen so only cells which are specified by lack of morphogen will be observed
• No information so no pattern is developed

Question 11

Explain the cell fate pattern where the morphogen is expressed uniformly

Answer 11

• Morphogen gradient does not change with a change in distance
• Cells will all have the same fate as all exposed to the same concentration of morphogen
• Information is the same so no cell fate pattern is observed

Question 12

What 2 things must a morphogen do?

Answer 12

1. Induce different outputs at different concentrations

2. Act directly at a distance

Question 13

Explain how instructive signals are morphogens

Answer 13

The high concentration of morphogen induces a different output to lower concentration

Question 14

Explain how permissive signals are not morphogens

Answer 14

A signal permits cells to respond to another source of information perhaps from a previously inherited cell fate determinants

Question 15

Explain how providing a second source of the signal tests the hypothesis of morphogens as instructive and not permissive signals

Answer 15

• Using an ectopic morphogen
• Morphogen/instructive signal would result in mirror image of patterning
• Permissive signal would have no affect and show normal patterning of signal

Question 16

Explain how providing a signal at a uniform concentration tests the hypothesis that morphogens are instructive signals and not permissive

Answer 16

• Morphogen/instructive signal would result in cells at the same fate
• Permissive signal would show no effect as the effect is pre-determined

Question 17

Explain the bucket brigade morphogen theory compared to how morphogens actually act

Answer 17

Morphogen: can effect cells at a distance away from the source

Bucket brigade: cells provide information,ation to the adjacent cell which causes the adjacent cell to makes its own signal and provide information to the neighbouring cell to change its fate

Both theories suggest the first cell is responsible for patterning the field of cells. However, morphogen specific the fate of all cells, in the bucket brigade, each signal induces another

Question 18

Explain how using genetic engineering to make a morphogen a juxtacrine tests the hypothesis that morphogens acts at a distance and the bucket brigade is incorrect

Answer 18

• Restrict the morphogen movement so signal can only be received by adjacent cells
• Other cells would all assume the fate that would be observed if no morphogen was present
• Bucket brigade would not be affect and cells could still provide information to adjacent cells

Question 19

Explain how making a genetic mosaic that lacks the receptor for the first signal in one cell tests the hypothesis that morphogens acts at a distance and the bucket brigade is incorrect

Answer 19

• Make cell that lacks receptor for the morphogen released by the signalling cell
• The cell that lacks the receptor doesn’t specify to its normal fate as can’t ‘see’ the receptor
• The gradient still forms and doesn’t impact other cells so they still specify as normal
• Bucket brigade theory wouldn’t be affected by this

Question 20

Is passive diffusion what establishes a morphogen gradient?

Answer 20

• Passive diffusion would only generate a shallow gradient and be inefficient

Question 21

How are steeper morphogen gradients established?

Answer 21

• Binding to a molecules in the ECM (e.g HSPGs) and high concentrations of receptor can generate a steep gradient called restricted diffusion
• Rapid degradation of the signal in the extracellular space may also generate a steep gradient

Question 22

What are HSPGs and what is their involvement in signalling?

Answer 22

• HSPGs are found in the ECM and bind to ligands

* Regulate diffusion by sequestration or slowing and facilitating diffusion

Question 23

How does planar transcytosis help establish morphogen gradients?

Answer 23

• A pit forms in the cell membrane and engulfs the morphogen in a vesicle
• Repeated cycles of endocytosis and re-secretion through the cells allows certain morphogens to travel through the cell in a tissue and restricts its movement

Question 24

What evidence is there for transcytosis in Dpp (TGFbeta) signalling

Answer 24

Antibody staining shows that Dpp is found in vesicles and mutations that block vesicle formation cause Dpp to act in a juxtracrine manner

Question 25

What factors help establish a morphogen gradient?

Answer 25

1. HSPGs
2. Planar transcytosis
3. Timing
4. Rapid degradation of the signal in the extracellular space
5. High concentrations of receptors

Question 26

How does timing help establish morphogen gradients?

Answer 26

• The cells need to know when they should be reading the concentration of morphogen
• As the gradient is established, gene expression is changing with time
• There must be a mechanism to block premature specification. The cell probably waits for the steady state of receptor activation to be achieved- check point mechanism is poorly understood

Question 27

What is the steady state of receptor activation?

Answer 27

The rate of degradation of secreted ligand is balanced by the rate of which new ligand is being produced

Question 28

What model is used to describe how cells read or interpret a grained to make a cell fate decision?

Answer 28

Transcriptional read-out model

Question 29

Explain the transcriptional read-out model

Answer 29

o High concentration of morphogen often results in higher concentration of an activated transcription factor

o Receptor activation causes TF to enter the nucleus and direct transcription

o It is the same TF in every cell (i.e same pathway/receptor/ligand)- these cells are initially identical

o Can convert the activity of an extracellular ligand to the activity of a nuclear localised TF

Question 30

Explain how the transcriptional read-out model can be seen experimentally with Bicoid

Answer 30

1. Bicoid is a morphogen and a TF
2. Bicoid mRNA is localised at the anterior of the egg and is translated into a protein during early embryogenesis
3. Bicoid protein then diffuses through the cytoplasm and accumulates in nuclei of the syncytial blastoderm generating a concentration gradient

Question 31

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

Answer 31

Positive feedback

Question 32

How is TF concentration interpreted at the DNA level?

Answer 32

o Enhancers that regulate one cell fate genes have lower affinity for the TF than those of a different cell fates

o At medium TF concentrations: even though both sets of genes see the same levels of TFs, only the high affinity enhancers can bind enough TF to activate gene expression of the cell with higher affinity

o Result: cell genes with higher affinity are activated

At high TF concentrations: both cell genes are activated as there is enough TF for binding in both the high affinity enhancer and low affinity enhancers

Question 33

How does the affinity affect the TF + DNA ⇌ TF:DNA reaction?

Answer 33

o TF + DNA ⇌ TF:DNA (TF bound to DNA)

o Normal affinity binding: reaction is at equilibrium

o High affinity DNA binding: forward reaction is stronger, reverse reaction weaker. More TF:DNA (forward reaction favoured)

o Low affinity DNA binding: reverse reaction is stronger than forward reaction (reverse reaction favoured). Less TF:DNA, more TF + DNA.

Question 34

What is cross talk regulation?

Answer 34

High morphogens switch on both cell genes, but one cell that encodes a repressor/switches off the genes of another cell

This is why cells don’t all have the same characteristics