Chick Flashcards
before the limb grows, what is the first thing to develop?
mesenchymal cells forming a limb bud
how did saunders find the ZPA?
he took a bit of the bud on the posterior to the anterior but not the other way around, he got dramatic duplication of the limb as a mirror reflection - you see that you have a mirror go 4,3,2
what happens when label the ZPA mesenchymal tissue that you transplant when you place it on the anterior of the limb bud?
- you see that the digits are derived from the host cells not from the ZPA graft
depending on what time you transplant the ZPA, what do you get in terms of from early to late?
- the earlier the transplant, the more proximal duplication you get. For example if you do it very early you get a duplication of the radius and ulner or even earlier you can get an entire limb. later transplants only duplicate the limb buds as a mirror at the 2 digit
what did the transplant of the ZPA demonstrate?
that the ZPA was secreting something which was telling the limb bud mesenchyme, where there were on the limb bud
what is the french flag model?
imagine there is a french flag, and there is a gradient from the blue end and a sink at the red end. The cells use this gradient to determine where they are in the flag depending on whether they are within the blue con, white conc or red conc
what is a morphogen?
a substance whose concentration determines cell fate- it must have at least 2 threshold- specify at least three fates
what should happen if you put less of the ZPA than is present on the posterior? describe in relation to morphogen gradient?
you should get only the middle part of the mirror reflection- as there will a lower amount of morphogen on the anterior side you only get those digits that are encoded by a lower amount of morphogen on the posterior side
what would happen if you transplanted a ZPA closer to the mid point of the limb bud on the anterior side?
you will never get a 3 digit because the concentration at the tip of the limb bud is receiving too much morpogen to get a low enough concentration
how was RA first implicated as the morphogen?
they found that there was a high concentration of retinoic at the posterior and a low at the anterior so they thought this may be the moprhgen acting from the ZPA
how can you show that RA is ‘sufficient” to induce a ZPA?
if you take a bead and soak it in RA and transplant it- it can act as a ZPA and induce a mirror image of the digits
what was RA shown to do?
it was shown to induce Shh in the limb bud which is then the morphogen
what was found to be the morphogen acting in the ZPA?
shh
how was RA disproved at the morphogen?
?? (maybe you can’t rescue Shh with RA) if you tranplant RA but use MO against shh then you won’t get an effect
what are the three things that Shh was shown to do which allowed it to be recognised as the morphogen?
- in situ showed that it is expressed in the ZPA region very distinctly
- misexpression by placing it in the anterior causes a limb dupliacatiom, loss of function leads to missing posterior structures
how was shh first discovered as a potential ZPA morhphgen?
people were cloning Shh and did an insitu and found it was expressed in the limb bud
how was the apical ectodermal ride first implicated in the limb bud development?
someone peeled off the AER and you se that there is extreme truncation
what happens when you removes the ridge early compared to when you remove it late?
you get more proximal truncation the early you remove it
what does the fact early removal of the AER vs late removal of the AER suggest about the development of the limb?
the development occurs in a sequence in which the proximal cells re patterned first and then the more distal later
how was FGF first implicated as working in the AER?
if you remove the AER and add FGF, you dont get truncation and get a fairly normal limb bud
once you have implicated gene such as FGF and SHH, what is the next step to prove they are relevant ?
- knock out the expression and see if it affects the process of interest
how was FGF shown to control limb bud formation and initiate it?
if you soak a bead in FGF and insert it either close to a leg or a wing, then you get one of the relative types of limbs from new.
what is the progress zone model?
beneath the AER- there are dividing cells and as the limb grows, the descendents of these cells get left behind and left in the proximal and as the AER moves away they start to differentiate and those that divide from the AER get more distal fates depending on how late they divide. So to do this the cells in the yellow zone, the dividing cells, measure how long they have been in that zone: short time= proximal and long time= distal fat
how was the progress zone model attemptivley proven?
by irradiating the cell in the proliferating zone to stop them going into the cell cycle and that truncation occurred depending on when you irradiated
what would be a good way to test whether the time that a cell spends dividing determines its fate?
you can take the cells out, allow them to divide and then if you put them into a young limb bud, do they grow older tissue or earlier tissue
how was the principle of non-equivalence produced? what does this tell you?
they took materiaal from the middle of the mesenchyme of the leg and planted it in the wing at various times- they took proximal limb tissue and implant it under the AER- you get tissue that is like the limb from which you took it- but the position of the tissue is like where it was implanted. this tells you that the decision between being leg or being wing, is decided before the decision to be proximal or distal and that the signals for proximal and distal are the same in the leg and the wing.
how id the dorsal-ventral patterning generally thought to occur?
dorsal and ventral ectoderm may be separated by ‘lineage restrictions’ very early in development- this is apparent from a very early stage. the daughter cells on the dorsal and ventral do not appear to cross a boundary. Wnt7a is necessary and sufficient to doornails the limb. Wnt7a loss causes the dorsal side of the limb to become the ventral side and causes missing posterior digits, Wnt7a induces the lmx-1 to be expressed on the dorsal side- mice lacking lmx-1 produce ventral skin on both sides of the paws. engariled-1 represses the dorsalising effect of wnt-7a in the ventral side for the limb
how frequently does a new somite form?
roughly every 90 minutes
how many pairs of somites are there in the chick?
55 pairs
how was claudio first drawn to looking at he relationship between somites and nerves and how did he first investigate?
he wondered how nerves and somites were related to each other in terms segmentation and so he stained the nerves and found that they only went in the rostral half of the somite so he then wanted to see if this was a property of the nervous system or somite
how did they look into whether it was a property of the nervous system or somites that induced the nerves to go through only the posterior of the segment?
they took out a piece of neural tube rotated it and then put it back in- if it was something in the neural tube and nerves that only allow nerves to come out of a certain spot then you should see nerves going through anterior now- but they found it was normal- so they then rotated the somite- they found that the nerves were going through the anterior now
what compoments are involved in the sending of the neural crest cells only through the posterior?
- Eph/ Ephrin
- semaphorins
- unknown protein that binds to peanut agglutinin
what is a growth cone collapse assay?
- you grow a growth con eneuron in vitro and apply the factor of interest- if it is inhibitory to the growth cone it will cause collapse
how did they show that the sclerotome causes axon collapse?
took took extract from sclerotome and cultured axons on- saw i stimulate collapse
how was the oscillations in the prismatic mesoderm first observed?
a guy did in situ for hairy1 expression in a chick embryo for the 15 somite stage and found that there was always three different expression patterns in the embryo- the white stripe lacking expression was moving. He then found at the 16s and 17s stage, the same pattern was seen
how was the different time expression patterns of hairy1 shown in the embryo? what did they find?
- they cut an embryo in half down the midline
- then they either fixed it straight away and did in situ or they waited and then did in situ after 30 minutes and so one. They found that after 90 minutes the 3 patterns start repeating themselves. this was called the segmentation clock
how can you observe the somatic clock?
- can’t in a chick because can’t do transgenics
- can produced a hairy1 - component transgenic and watch the waves in expression along the neural tube during somite formation
how many waves are thought to induce a somite?
10-12 waves before it becomes a somite
what is the clock thought to be
the clock is a cell autonomous oscillator which drives notch signalling and very 90 minutes it goes through a cycle- yeah neighbour is a bit out of phase and at the same time the there is a wave of FGF which gates it, all the cells that are approximately synchronous “see” the wave pass and as it passes at a particular point now all of the cells similar in the wave time produce a somite.
how was the wave clock model shown to be wrong ?
- the clock wave models suggests that there is a cell autonomous oscillation of notch and WNT signalling that acts last the cell clock. There is also a wave of expression which travels up the embryo and this is thought to be FGF. It is the interactions between these signals which is thought to determine the size and formation of the somites.
- however, sten et al showed that simply placing posterior primitive streak with noggin into he lateral place mesoderm is able to induce a synchronous cluster of somites that function normally. There is no oscillation in hairy-1 expression and there is no wave as they are in a cluster. This shows that the wave clock model is not required for somite formation.
- however it does seem to be needed for the patterning of the rostral and caudal ends of the somites
one you find that simply the inhibition of BMP can form somites, how do you proceed from there to determine whether these somites are forming sequentially and is there a clock?
You can make a movie and see that they all form at the same time - there is not clock
how can you test that the somites formed via BMP inhibit are actually somites?
used transgenic chips for GFP for the graft- put posterior primitive streak into the lateral plate mesoderm from a non GFP chick + noggin and you see that you get somites which express the somite marker (paraxis)- can put these somites into a normal embryo and they function normally (use GFP to identify donor and host)
how was the somites formed via BMP inhibition shown to have no charactertsics of hairy1 oscillation?
you can use in situ over time and you see that there is no change - no wavefront
