Sem 222222 Flashcards
Which of the three axis is established first in the embryo
A/P
Then L/R
Then D/V
Basic difference between specification and determination
Specification is where cells restrict their potential but this is not stable. It can be changed.
Determination is where their fate is set and can’t be changed.
When does most growth happen in development
What is accretion
Most tissues and organs are layed down early in development
In later stages there is a lot of growth.
Growth of ECM around the cell to give it a larger size.
Two models of embryonic development
Funnel-
The embryos are very similar in the early stages and they become less and less alike as development continues.
Hourglass-
The early stages of development are not that similar. The middle stages are similar and the final stages are not similar.
what is a morphogen and what does it do
how does the default fate occur
a soluble secreted molecule that acts at a distance to specify the fates of cells. it is instructive and tells the cell what to do.
it can specify more than one cell type by forming a concentration gradient
when no morphogen reaches a cell.
what happens if too much morphogen is made
what will an ectopic source of morphogen at the opposite side cause
all cells will take the fate caused by high morphogen concentration.
it will cause pattern duplication, the same fate pattern from both ends reached the middle.
How to distinguish between a morphogen and a permissive signal
ectopically express them.
morphogens expressed ectopically will change the nearby cells fate because they are instructive.
permissive signals will not change a cells fate because all they do is tell a cell to go and not what to do.
the concentration of morphogens is important and this is not the case for permssive signals where it will do the same thing no matter how much there is.
what is the bucket brigade and is this used by morphogens
one morphogen travels to a cell and causes it to release another morphogen to a further cell.
a morphogen is a single signal that induces many cells and so this is not used by them.
Testing for a morphogen by tethering it to the membrane
this means it can no longer diffuse.
true morphogens will only influence the cells touching it, any further cells will have the default fate.
the bucket brigade which is not done by morphogens would mean that the cells touching the morphogen would change fate, causing them to release a second signal that would affect further cells.
from this experiment we know it is a morphogen if only the touching cells are changed.
Testing for a morphogen by removing the membrane receptors
A true morphogen would cause the default fate if the receptors are removed.
Bucket brigade would not affect the cells because each cell is responding to a different signal from the one next to it. So the lack of receptor will not affect many cells that respond to all the different signals down the cascade.
How are morphogens able to create such a steep gradient 3
what is the name for this
they bind to molecules in the ECM such as HSPGs, many ligands have a HSPG binding site. they are sticky and will keep the ligands in an area for longer and also stop them straying to other areas, this is called restricted diffusion.
there are also areas with high levels of receptor which would mean that that area would have more signalling and this would help the gradient.
rapid degradation of the morphogen can also create a steep gradient, this is to stop all the cells having too much morphogen and all having the same fate.
what is planar transcytosis and what is it for
example
it helps to establish morphogen gradients
a pit forms in the cell membrane and engulfs the morphogen in a vesicle which travels across the cell and is spat out the other side, it is then taken up by the next cell.
antibody stains show that dpp is found in vesicles and mutations that block vesicle formation cause dpp to act in a juxtacrine manner and only work on neighboring cells.
Why is premature specification a problem
it takes time for the morphogen to be made and for it to reach cells.
at first cells will see a low conc of morphogen and they could start to specify now, but they wait for a constant level to be reached so that they dont take on the wrong fate too early.
what is the transcription read out model
bicoid example
higher conc or morphogen means higher conc of activated TF.
this will cause more or less transcription which will lead to a different fate.
in a drosophisl oocyte, bicoid mRNA is localised at the anterior of the egg and is translated into protein during embryoogenesis.
bicoid protein diffueses through the cytosol and accumulates the in the nuclei making a concentration gradient.
what determines how long a TF is bound to the DNA sequence.
what does longer binding cause
some sequences have a higher affinity for the TF than others.
some will stay bound for longer to higher affinity sequences and this will cause more transcription
How will the promoters differ between cells close to the morphogen source and cells far from it.
cells far from the morphogen will need higher affinity promoters because hardly any morphogen will activate TFs. the few that do bind will have to stick for a while to activate cell fate.
cells close to the morphogen will have low affinity promoters so they need a high level of TF. because they wont stick to the promoters for long. this is good because it stops them from being activated by low morphogen concentrations.
the closer the cell to the morphogen the lower the promoter affinity.
How do cells close to the morphogen not have all cell fates activated because they are exposed to all levels of morphogen
this is topped by having the low affinity promotors produce a repressor for the high affinity promotors. meaning only one cell fate is activated.
this type of regulation is called cross talk.
the high morphogen concs will activate all the promoters but some genes will repress others when they are activated.
what are the hh and wnt genes involved in
patterning many tissues and developing organs
regulation of stem cell fate and cell division and in the maintenance and initiation of cancer.
how was hh discovered and what does the hh mutant do
the hh mutant will have a lack of naked cuticles and just all the denticles lined up
it was disocvered in the 1980s by nusslein volhard
how are hh and wg linked
what are their mutants like
they are in a mutual feedback loop
hh is required for wg expression and wg is required for hh expression.
this means that the wg and hh mutants will look the same because if one is lost then so is the other one.
how was wg discovered
it was discovered in mice and was called int1
when they discovered that int1 and wg were the same gene they called it wnt.
what are the vertebrate homologues of hh
what other wg homologues are there
shh, desert and indian hh
drosophila have 7. there are 18 in invertebrates
c elegans has no wg.
hh formation and modifcations
how does the resulting insoluble protein leave the cell.
the hh gene is translated into a protein that has an N terminal signal sequence that targets them to the secretory pathway.
after it has entered the pathway the signal sequence is cleaved off.
there is also an autoproteolytic domain that cleaves itself off the protein.
then there is a cholesterol modification to the C terminal, then there is palmitoylation which is the addition of the fatty acid group to the N terminus.
PHOTO
this results in an insoluble protein that cannot leave the cell. to change this the dispatch gene (12 domains) and scube proteins in vertebrates help to allow hh to leave the membrane but we dont know how.
they could help load hh onto lipoproteins which can diffuse away or they could use HSPGs.
wnt formation and modifications
the gene is translated into a protein that has an N terminal signal sequence that targets it to the secretory pathway and this is cleaved off after it enters the pathway.
it then has two modifications, one is palmitoylation and the other is palmitoic acid.
this will make it insoluble and again this might be overcome by lipoproteins or HSPGs.
or the wntless transmembrane protein maybe be involved in getting wnt to the membrane and allowing it to diffuse away which needs HSPGs.
what are cytonemes and what do they do
they are long protrusions off a cell
signalling molecules such as wnt accumulate at the tips of the protrusions
the tip will reach out and touch another cell and induce a signal in the receiving cell.
the hh signalling pathway
what does ptc do and what is the stiochemistry
ptc is membrane bound and (12 domains) binds to the hh ligand.
when hh is not bound ptc inhibits smo.
ptc and smo dont work in a one to one stiochemistry, a single ptc can inhinit a large number of smo molecules.
ptc regulates the subcellular localisation and stability of smo, ptc can stop smo from getting to the cell membrane.
when hh binds to ptc they both get internalised and degraded and smo can now reach the cell membrane surface. smo can now accumulate at the surface and the hh pathway is activated.
How are the cells different in mammals when the hh pathway is activated
cillia on the cell are used as antennas
when there is no hh , ptc is accumuated on the cell membrane.
when there is hh there will be smo accumualtion on the membrane because all the ptc will be internalized and degraded.
how are the hh genes activated and repressed
ci/gli the TF drives transcription of the hh genes.
without hh, ci is kept out of the nucleus by two complexes-
1- cos2 (a scaffold kinase) and fused (a serine threonine kinase).
2- sufu (supressor of fused) which is a cytoplasmic ci anchor
these complexes can be bound by three other kinases (CKI, PKA, GSK3). these will phosphorylate ci and cause it to be recognised by a ubiqutin ligase called slimb which will partially break it down, this will form a smaller active version of ci which is a transcriptional repressor called cir.
when there is hh present the phosphoylation of ci is blocked and so ci is not degraded and it can enter the nucleus activate the hh genes.
hh signalling and negative feedback
the more ptc there is the more smo is repressed and so less hh genes are activated and less ptc is made.
so ptc inhibits its own signal and this limits the level of activation.
in vertebrates what cant happen to gli in the hh pathway
it cannot be proteolysed into a repressor so it is always an activator and can make more ptc and this is a feedf foreward response.
