MBB6346 Genetic pathways from zygote to organism Flashcards Preview

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How can asymmetric division occur in response to intrinsic factors?

- A factor within the maternal cell is asymmetrically distributed before division which is then inherited asymmetrically between the two daughter cells
- This then drives the change in cell fate


How can asymmetric division occur in response to extrinsic factors?

- The two daughter cells encounter different environmental signals which change their fate.
- The cells respond differently to signals. This could be due to polarity in the system (gradients) or due to the cells differing competence (intrinsic)
- This is often associated with tissues


What is a morphogen?

A molecule that acts in a concentration dependant manner to determine cell fate


What can a morphogen be?

- Does not have to be a transcription factor just has to be able to activate a signalling cascade
- It can also be a metabolite or an extracellular peptide


What is meant by a cell being competent to perceive a morphogen?

- Morphogens must be capable of being perceived by cells to elicit a response
- A cell must be able to respond to it through the correct receptors/transporters


Is it just the morphogen concentration that decides morphogen activity?

- It is the overall output
- If there is a morphogen inhibitor present then this will affect the overall morphogen activity and change the cell fate


Why are C. Elegans a major model organism used in developmental biology?

The complete lineage of all cells has been mapped


Outline the first divisions of the C. Elegans zygote?

- The C. elegans’ zygote, after fertilisation, undergoes an asymmetric division to give AB and P1 daughter cells.
- AB gives rise to somatic cells, in particular, the hypodermis and neurons, while P1 gives rise to the germ line in one division path and muscles and gut in another. P lineage cells are germline cells.


What are the cell fate determinants in the C.elegans zygote?

The cell fate determinate for AB is MEX5 and for P1 is PIE-1


How do MEX5 and PIE-1 cause asymmetric division?

- These are both maternal transcripts, already present in the maternal egg
- The proteins become polarised before the asymmetric division occurs causing preferential inheritance. Once the division happens, they are segregated into AB and P1


What are PAR proteins?

PAR proteins are a mix of membrane binding proteins, kinases and components of the ubiquitin degradation pathway.


Outline the PAR distribution in the unfertilised c.elegans zygote

- In the unfertilised egg, PAR proteins are mostly associated with either the cell membrane or the cytoplasm. The PARs associated with the membrane stop the other PARs from binding there.


What is the key driver of polarity in the C.elegan zygote?

- The key driver of polarity here is sperm entry.


How does sperm entry drive polarity in the C.elegans zygote?

- The sperm entry point becomes the posterior pole of the egg and causes changes in the membrane, resulting in some PAR proteins shifting to the anterior and the others then moving onto the posterior membrane.
- Interactions between the PARs stabilise this localisation. This localisation is crucial because these posterior PAR proteins act on MEX-5 and this results in MEX-5 being localised to the anterior pole.
- It is MEX5 that drives polarisation of PIE-1 as MEX5 inhibits PIE-1.


Give evidence for the intrinsic control of c elegant zygote asymmetric division

Laufer et al
- The lab specifically burst cells within the egg to look at the effect of the remaining cells.
- They burst the AB cell, leaving the P1 cell. The cell continues to divide and they followed the division. They saw that the lineage is the same as that in the Wt
- This tells us that intrinsic factors driving the division because otherwise the removal of the AB cell would affect the linage of P1


How was the role of PIE-1 in the c.elegan zygote?

Mello et al, 1992
- Carried out a mutagenesis screen and found a maternal mutant PIE1. In these mutants, the complete germ line lineage is deleted but the gut and muscles still form.
- The maternal gene is present in the unfertilised egg and inhibits transcriptional elongation by preventing phosphorylation of the RNA pol II C-terminal domain by CDK9 and inhibits genes associated with somatic cell fate. This causes cells to be maintained as germline cells.
- Pes-1 is a marker of somatic cell fate and when stain in embryo can see it is not found where PIE-1 is found showing that PIE-1 drives germline fate


Give evidence for when PIE-1 localisation occurs in the embryo

Reese et al, 2000
- An embryo expressing PIE1-GFP was live imaged to show PIE-1 localisation in the posterior pole occurs before the asymmetric division
- PIE1 inhibits the regulation of somatic genes and its protein becomes polarised before asymmetric division


What kind of proteins are PIE-1 and MEX5?

Group of maternally inherited mRNAs encoding zinc finger RNA binding proteins


What is the phenotype of a MEX5 mutant?

- Disruption of mex-5 causes embryonic death with a terminal phenotype that includes proliferation of muscle (MEX = muscle excess)
- This is likely explained by cells being blocked in AB lineage fate and hence acquire P1 fate


Give evidence for the role of MEX-5 in fate determination

Schubert et al, 2000
- Can see before asymmetric division, MEX5 is polarised anteriorly as shown by the immunostaining.
- Investigated the localisation of PIE-1 in the Wt and MEX5 mutant embryo. In the WT that these proteins are confined to the germline P2 cell but, in a MEX-5 mutant, their expression is now found throughout the embryo
- MEX5 inhibits the expression of the germ line promotors in somatic cells – negatively regulating PIE1


How do we know that MEX5 does not TRANSCRIPTIONALLY regulate PIE1?

- We’ve seen that PIE-1 can inhibit the transcription of some genes. However, PIE-1 are maternal transcripts so the transcripts are already in the embryo.
- Therefore, MEX5 does not transcriptionally regulate PIE1 as it is the protein that is localised posteriorly not the RNA.


How does MEX5 regulate PIE1?

DeRenzo et al, 2003
- A yeast-2-hybrid screen for interacting partners of MEX-5 and PIE-1 identified ZIF-1. Further interaction studies found that ZIF-1 is part of an E3 ubiquitin ligase complex that targets proteins for breakdown ZIF1 is an E3 ubiquitin ligase and targets proteins for degradation
- In RNAi of Zif1, PIE1 is found in all the cells but in Wt it is localised.
- MEX5 recruits ZIF1 to target PIE1 and to cause its degradation anteriorly.


When is polarity determined in the c elegant zygote?

Polarity is beginning to be determined very early before polarisation of MEX5 and PIE1


When are PAR proteins localised in the c elegant zygote?

Kemphues et al, 1988
- PAR mutants found in maternal screen looking for mutants that effect polarity determination
- PAR1 and 2 are localised posterior cortex before division while PAR3,4 and PKC-3 are present in the anterior cortex.
- PAR proteins show polarity


How do PAR proteins become polarised?

Goldtsein and Hird, 1996
- The sperm entry point defines the posterior pole
- Manipulated sperm entry so it occurs anteriorly. This reverses the polarity of the zygote
- Experiments were also done examining what happens if the sperm enters at a lateral position and in these instances, the sperm pronucleus quickly migrated to the nearest pole and this became the posterior. Cytochalasin D treatment inhibited this movement indicating that intact actin microfilaments are needed for this process.
- Sperm donates a centrosome to the entry point (contains actin) affecting actin within the oocyte


What is meant by the fountain head streaming affect?

- The mesh of actin is contracting and relaxing and sperm entry results in relaxation at the posterior pole meaning actin only contracts at the anterior. This forces cytoplasm to move to the other end. This is known as a fountain head streaming affect.
- This is critical for the localisation of PAR proteins


Outline the PAR distribution in the unfertilised c.elegan zygote

Before fertilisation, PAR3, PAR6 and PKC-3 are present around the entire cortex. These proteins inhibit PAR1 and PAR2 from the cortex


Outline the PAR distribution in the fertilised c.elegan zygote

- After fertilisation, and fountain head streaming occurs, PAR3 and 6 are driven into the anterior pole allowing PAR1 and PAR2 from the cytoplasm to bind to the membrane in the posterior pole.
- This results in PAR3 and 6 localised at the anterior cortex and PAR1 and 2 and the posterior cortex. The PAR proteins are mutually exclusive.


How are PAR proteins mutually exclusive?

PKC-3 phosphorylation inhibits their ability to bind the anterior region by phosphorylating PAR-1 and PAR-2. In turn, PAR-1 inhibits PAR-6/6/PKC-3 from binding the posterior region by phosphorylating PAR-3, whilst PAR-2 can inhibit PKC-3.


How does PAR distribution affect MEX5 polarisation?

- PAR1 acts on MEX5 causing its dissociation from proteins and RNA. Usually it is associated with protein and RNA making it slow moving.
- Once PAR1 causes its dissociation, it produced two pools of MEX5 – one bound to proteins which is slow moving and another which is free from proteins and fast moving.
- MEX5 is unable to bind proteins in the posterior due to the presence of PAR1 and its subsequent phosphorylation meaning MEX5 moves anteriorly where it can bind proteins and become slow moving again.
- This results in MEX5 to gradually localise anteriorly