Lecture 4

Question 1

How can you experimentally modify a potential morphogenic molecule to make it juxtacrine

Answer 1

Using genetic engineering to add a transmembrane domain region to the protein sequence to make it a membrane bound signal

Question 2

How may it be possible to achieve a straight linear morphogen gradient

Answer 2

Active movement of the morphogen may achieve a linear gradient

Question 3

How can morphogens move through cells in a tissue and give an example of a molecule for which this is the cane

Answer 3

Planar transcytosis - Repeated cycles of endocytosis and re-secretion. Dpp is transcytosed and antibody staining has revealed its presence in vesicles

Question 4

The shape of the morphogen gradient is what encodes the information to direct cell differentiation, T or F

Answer 4

T

Question 5

What would be seen in a field of cells as a result of ectopic grafting of another source if the secreted factor was a morphogen

Answer 5

A mirror image of cell differentiation would be seen

Question 6

Explain what is meant by the bucket brigade signalling mechanism and why this is therefore not used by morphogens

Answer 6

The bucket brigade mechanisms uses sequential signalling of the cells that acts immediately on the adjacent cell. Expression of one signal induces a target cells to produce another different signal. This signal acts on the next cell in the sequence to induce production/secretion of another different factor and so on. As this signalling occurs between adjacent cells and not over distance, this isn’t a type of morphogen signalling

Question 7

Explain how a cell interprets a morphogen gradient before responding and determining its fate

Answer 7

Morphogen concentration is directly correlated to the activation of transcription factors inside the cells. Higher concentration of morphogen often results in a higher concentration of an activated transcription factor. In this model, receptor activation causes transcription factors to enter the nucleus and direct transcription. Levels of activated transcription factor determine the fate of the cell

Question 8

How can a higher effective concentration of a morphogen be achieved

Answer 8

Preventing the morphogen from diffusing in axes that aren’t desirable

Question 9

What would be the effects of making a morphogen juxtacrine and how would this differ if the signal was part of a bucket brigade signalling mechanism

Answer 9

If the ligand was a morphogen, making it juxtacrine would lead to only the first cell being induced to adopt its fate as the ligand will be unable to act at a distance and direct differentiation by diffusion. This would be due to it becoming membrane bound and only able to act on adjacent cells. If the factor was part of a bucket brigade mechanism, then the signalling would be unaffected as adjacent cells would still receive their ligand and thus go onto produce the next sequential secreted factor.

Question 10

Define a morphogen

Answer 10

A soluble secreted molecule that acts at a distance to specify the fates of cells

Question 11

How is the timing of morphogen gradient establishment critical to cell differentiation

Answer 11

The cells are prevented from responding at an inappropriate time where the morphogen concentration won’t have reached that required by the particular cell to direct its correct fate. This is likely achieved by the cell waiting for a steady state of receptor activation to be achieved but the molecular mechanism by which this occurs isn’t understood

Question 12

What are the two main attributes of a morphogen

Answer 12

Morphogens act at a distance to induce different output or cell fates at different concentrations by forming a gradient in the embryo.

Question 13

Explain how strict thresholds of cell differentiation are achieved even if the morphogen gradient isn’t steep

Answer 13

Positive feedback helps a cell commit to its specific fate. If one of the genes switched on by high affinity site binding encodes a transcription factor – it can, amongst other things, activate its own expression

Question 14

What happens at high transcription factor levels and how does this confer a particular cell fate

Answer 14

At high concentrations of the transcription factor the low affinity sites will now allow binding. Binding of the transcription factor to these sites will result in differential gene expression and a different cell fate. More ligand will increase activation of low affinity sites

Question 15

How would the bucket brigade signalling mechanism be affected by a lacking receptor

Answer 15

Cell fate wouldn’t be effected if the receptor knocked out from a cell was one for a ligand that acts upstream of the cell in the sequential signalling pathway. This is because at this point in the cell field, this ligand is no longer acting and instead a different ligand is acting on the target cell produce by the proximal adjacent cells. However, if the receptor knocked out was the first receptor in the sequential sequence then none of the cells would adopt their fate and would remain undifferentiatiated

Question 16

How do different levels of transcription factors dictate the different fates of cells under the influence of a morphogen

Answer 16

Binding of a transcription factor to the promoter seuequence of genes that confer a particular cell fate is an equilibrium reaction with an on and off rate. The strength of the equilibrium that’s moving gene expression towards its on state will control how strongly the gene is expressed. Increasing transcription concentration factor would increase on state by shifting the equilibrium to the right to counteract this increase in transcription factor levels. The concentration of activated transcription factor determines if it binds to high or low affinity sites. Each of these sites dictate a different cell fate and differential gene expression

Question 17

Why are morphogen gradients usually exponential decays

Answer 17

Morphogens move by passive diffusion throughout the embryo leading to the exponential decay appearance of the gradient

Question 18

Give an example of an experiment that has proved a gene product is a morphogen by ectopic grafting

Answer 18

Ectopic grafting of an shh soaked bead opposite to its normal site in the chick limb bud leads wing development that defined by a mirror image duplication of the digits etc.

Question 19

What would be the effects of forcing a uniform gradient of a secreted factor across a field of cells if this factor was a permissive signal

Answer 19

There would be no abnormal effects on cell development. The same number of fates will be produced and each cell would adopt the correct fate

Question 20

How is higher information encoding achieved by morphogens

Answer 20

More information is encoded by the higher morphogen concentrations closer to the source

Question 21

Instructive signals are morphogens, T or F

Answer 21

T

Question 22

All molecule involved in patterning of the embryo are morphogens, T or F

Answer 22

F – permissive signals aren’t morphogens

Question 23

How do the low affinity sites allow differential gene expression if the high affinity transcription factor binding site are also being activated

Answer 23

One of the genes switched on by transcription factor binding to the low affinity sites will turn off by transcriptional repression) the high affinity genes in a process called crosstalk

Question 24

What would be the effects of forcing a uniform gradient of a secreted factor across a field of cells if this factor was an instructive signal

Answer 24

This would result on the field of cells only adopting one fate as all cells would receive the same ligand concentration

Question 25

What two mechanisms can account for the different cell fates achieved by morphogens

Answer 25

Temporal diffusion of the secreted signals from the source to where it is destroyed/sunk. Similarly, the magnitude of the morphogen concentration also causes the acquisition of different cell fates

Question 26

Shallowing morphogen gradients would encode far less information than steeper ones, T or F

Answer 26

T

Question 27

Heparan sulphate proteoglycans are sometimes referred to as co-receptors and bind many different ligands, T or F

Answer 27

T

Question 28

What happens at low-medium transcription factor levels and how does this confer a particular cell fate

Answer 28

At low-medium concentration of the transcription factor only the high affinity sites will elicit binding which will direct a particular cell fate and specific subset of gene expression

Question 29

How can non-beneficial morphogen diffusion be prevented

Answer 29

Morphogen binding to molecules in the extracellular matrix such as heparan sulphate proteoglycans allows sequestration and facilitation of morphogen diffusion, effectively increasing the size/steepness of the gradient in desirable planes. This acts to increase the amount of information encoded by the morphogen gradient

Question 30

What would be seen in a field of cells as a result of ectopic grafting of another source if the secreted factor was a permissive signal

Answer 30

This would have no effect on cell differentiation, all cells in the field would develop normally as the permissive signal only enables the cells to respond to a morphogen

Question 31

Why aren’t permissive signals morphogens

Answer 31

Permissive signals only direct a cell to response to an instructive signal

Question 32

How can rapid degradation of the morphogen at the sink increase information encoding

Answer 32

Increases the steepness of the morphogen gradient

Question 33

Decreasing the range of the morphogen gradient can be achieved by localisation of the morphogen in the desired range, T or F

Answer 33

F – this acts to extend the range

Question 34

Which morphogen is the only known one which itself is a transcription factor and how does it act

Answer 34

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

Question 35

How would creation of a genetic mosaic lacking a morphogen receptor influence differentiation

Answer 35

The cell lacking the receptor would adopt the same fate as the terminal cell in the field would normally. This would be due to the fact that the terminal cell usually receives the lowest/no morphogen signal. Cells after the one lacking the morphogen receptor would however development normally