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
what is the evidence for there being no wavefront model for determining somite size etc?
there is just a mass of cells- no line- not in a wave
what is the one property that the somites formed in the lateral plate mesoderm, are lacking compared to normal somites? how is this shown? what does this suggest about the wavefront model?
- rostral and caudal polarity- markers hairy1 and 2 are markers for the posterior part of the somite and they are not expressed - eph4 is a posterior marker and this isn’t expressed. This suggests that the wave front model is involved in deterring the anterior and posterior polarity of the somites
so if the segmentation clock does not control somite size, what could?
Hypothesis: somite size and shape is regulated entirely by local cell-cell interactions, e.g. constraints to cell packing:Embryological experiments suggest that somites are self-organizing structures, regulated by intrinsic properties of the cells and packing constraints for cells undergoing mesenchymal-to-epithelial conversion as they form spheres. We tested this in, with the following assumptions: (i) A cell mass is exposed to Noggin evenly and simultaneously; (ii) in response, cells polarize and elongate; (iii) polarized cells secrete extracellular matrix; (iv) polarized cells have to be exposed to extracellular space at both their apical and basal surfaces; (v) tight junctions form at the apical ends; and (vi) misplaced cells rearrange their polarity and attach to their appropriate ends. This causes cells to become arranged in spherical masses around a lumen. After a transition period of intense cell rearrangement, the somites stabilize. The number of cells they contain is relatively invariant, and their structure is similar to that seen in vivo. There is no tendency to merge into a giant structure, nor do very small stable somites form. Inhibition of BMP by Noggin may be a trigger for this conversion, consistent with the abnormal somite formation in Noggin-null mice (29), and may also “freeze” molecular determinants of axial identity (Hox code).

what is the F molecule idea?
every cell in the early embryo has something asymmetric in it for example and that this thing is F shaped- all you need to do is to tell the embryo which is the head and which is the tail and now you give it left and right
how was the F molecule idea first supported?
student was told to do in situ with shh and see where it was expressed in the gastrula- he showed it to another group and they found that activation RIIa was expressed on the right side too. Nodal was found to be expressed on the left of the node
after they had found that activity was expressed on the right to the node, how did they do follow up experiments? (2) what is the interaction between activin, nodal and shh?
they placed an active bead on the left and found that you could suppress shh on the left and turn off the nodal too
- then they tried the same misimpression with shh and put it on the right on a bead and found it induced nodal
- activin inhibits the expression of shh and Nodal and Shh activates nodal
after they found activin, nodal and shh to be expressed asymmertically in the node, what were the other 3 that they found ?
- they found pitx2 and cerberus on the left and FGF8 on the right and found an epistatic relationship between them
in terms of left right patterning, what two genes are consistently on the left in all species?
nodal and pitx2
what is pitx2?
a TF that confers leftness
what is nodal?
a left handed gene which controls pitx2 expression
what LR mouse mutants are there and what is involved? what human syndrome does this link to? what does this relate to in terms of LR patterning processes?
- there is a situs invertus mouse that encodes left-right dynein which is in cilia
- Kartegener’s syndrome indluces a defetc in ciliary beating too
- the node in the mouse has cilia which generate a flow of fluid to the left side
how can you test whether the nodal flow in the mouse is what is inducing LR asymmetry?
you can take an inverses viscerum mutant embryo and put it in a chamber which generates a synthetic flow and you can rescue the normal left to right polarity - these waft the Shh to the left??
what is strange about the process which is the symmetry breaking step?
it appears to be different in every single organism - but they all converge on nodal and pitx2
what is the most powerful thin that you can do in the chick?
can control the expression of genes and the labelling of genes in space and in time using electroporations and some other etchqniues
what is the knockdown technique used in chick ?
morpholinos
what is the benefit of cloning many species of birds?
- this allows you good evolutionary comparison because the species will be fairly similar in respects other than the distinct differences
- can look at molecular relationships
- can find things that are conserved across the genomes that you wouldn’t find when comparing distant species
how does electroporation work?
you can introduce DNA construct into a tissue and introduce it into a specific cell or tissue by an electric current and when it gets into the cell it will make RNA for you coding sequence or make protein- you can also crispr drivers etc, fluourascene labelled MO which will target the translation start its of mRNA. the placement of the neg and positive electrode determines where your DNA will go
how can using a fluorescein-labelled morpholine be used?
- you can insert the DNA and then you an anti-fluorescein antibody to label where you introduced the cells, then you can so an in situ for the gene that you hope to have been knocked down or over expressed and see the effects.
when you are using electroporation, how do you know which cells to target in the embryo in order to target a certain tissue or organ?
you know because of your fate map that you have constructed
how can you try to work out which enhancer regions of a gene are important ant for your tissue of interest?
you use an enhancer of your gene of interest upstream of a minimal promoter such as TK promoter- then you have GFP downstream- then you electroporate in to the entire embryo and see where your gene is expressed by filming it- if it is where you wanted then you know your enhancer that you inserted was correct
what is the TK promoter?
a promoter that will bind RNA polymerase but give you very low expression levels
why would you want to do an experiment where you have two different colour reporters with two different enhancers of a single gene?
if you inject both of these into an entire embryo, this will tell you the time and in the embryo development, in which each other enhancers is active
in addition to GFP, what other reporter can you use?
luciferase- emits photons
how is gastrulation viewed in the chick?
a flat disc can be seen with a primitive streak and a notochord
what is the pattern of goosecoid in the late gastrulae? (primitive streak stage)
the goosecoid is expressed in the node
how do cells move during the primitive streak stage of gastrulation?
the cells move around the embryo and then up through the primitive streak, through the node and then out again
what is the difference between a cell state and cell identityy?
a cell state changes dpending on its gene expression but a cell’s identity doesn’t change
where is goosecoid expressed in the blastoderm stage compared to the primitive streak stage?
- it is expressed in a small area in the early stage but in hensen’s node in the primitive streak stage
what are the dynamics of the goosecoid expression within the node during gastrulation?
there is a small subset of cells that do not move and signal to the changing cells that pass into the node to express goosecoid
how can you test whether cells expressing goosecoid in the blastoderm stage, are the same cells that express goosecoid in the primitive streak stage? what did they find?
use a fluo dye (DiI) that you can inject into certain cells, then you then let the embryo grow to the next stage and then fix the embryo, precipitate the DiI which comes out brown and then do in situ for gene if interest- the the cells are the same then they should have both labels. they found that in the top region- they are mostly blue cells but there are brown cells too which was in the deeper layers
how can you show whether goosecoid expressing cells induce other cells to express goosecoid?
you can graft goosecoid expressing cells fro a quail into a chick edge of the lateral plate mesoderm and ten you can see that a new primitive streak is formed and use in situ with chick-specific goosecoid prove- see that the chick cells have been induced
how can you sue microscopy to look at the movement of cells?
you can label nuclei and just follow the cell movement: couple GFP to histone2B- localising GFP to nucleus
how can you induce GFP nucleus labelling?
couple GFP to histone2B- localising GFP to nucleus
how can you study cell moments in gastrulation? what is found?
you can label nuclei and just follow the cell movement: couple GFP to histone2B- localising GFP to nucleus and then use microscope to film them. then you can triangulate the cells- you chose three cells and forma triangle and then follow the different cells over time. you look at whether the changed in shape of the lateral domain is different to cells contributing to early streak. transgress become long and thin in the middle - you see that cells movement via intercalation
what is the best way to view cell movement using reporters or cell labelling ?
do a moasic so you can distinguish the individual cells
what pathway is implicated in streak intercalation? how did they find this?
- non canonical WNt signalling pathway
they did a in situ for components of the PCP pathway such as flamingo, vangl1 etc and found them to be expressed n the posterior to the epiblast where the streak comes from
how can you test if the WNT non can pathway is involved in the cell intercalation? (2)
-inject a dnDSH which has a deletion of the DEP domain
via electroporation and inhibits signalling- inject a lot, in the construct you have GFP so you can trace. When you do this and use a mesoderm marker- instead of the mesoderm in a nice streak- you get a lump at the back- didn’t intercalate
- take three MO for the different genes involved in WNT intercalation and have a marker in the construct- you see the same as previous
- because you have used a marker for brachyury you have shown that the identity of primitive streak has not changed but that the different lies in a lack of intercalation
how can you test whether the driving of intercalation via non canon WNT signalling is sufficient to mediate all of the movements iwhtin the gastrulating embryo? what is the result?
- use a computer model with 3 assumptions:
The epiblast is composed of cells 2. The epiblast is under tension
3. The epiblast is a tight epithelium
4. cells have a preferred direction of intercalation in the streak-forming region
you find that it mimics primitive streak formation really well

what is a high epithelium?
displacing cells cause space around other cells so the rest of the cells move in to the space made- cells move less far away
how can they test the theory that call that is required for the primitive streak cell movement is for the PCP pathway to be stimulated?
they can inhibit PCP and mark the expression of a morpholino of 3 of the components of PCP signalling. The you just use DiI to mark cells in the plane of the gastrula as a way to measure their movements. Then you can watch what happens
- you can also make a model of the same thing
- does the model show the same thing as the embryo?
- you see the cells just go straight down and dont go up the midline they just go through the streak rather than up the streak - same in both models
what happens when you cut the gastrula into 4 quarters?
you get 4 complete embryos
what factor was first implicated as directing the movement of the intercalation in the primitive streak formation? how?
- cVG1 is expressed in the posterior marginal zone
- transfected COS cells with cVG1 construct to make them express it- grew them in culture and then implant them into the opposite side of the embryo to the PMZ - you see an entirely new axis from the host tissue
what is found if you put your cVG1 cells in the marginal zone compared to in the area pellucida?
you only get axis in the marginal zone and not in are pellucida
why did they look at WNT for the reason the axis is only formed when Vg1 is put in marginal zone?
because TGFbeta family normally acts with WNT
how did they test that WNT is involved in mediating the effects of cVG1 in the marginal zone (3)
- they did in situ and found it was expressed only in MZ, he then used the Vg1 with crescent- an inhibitor of WNT- you dont get an axis when transplanted in the marginal zone
- when you add WNT and VG1 to the area pellucida you get an ectopic axis
what is the key function of the hypoblast in early stages of development?
has an influence on polarity
how does the hypoblast general form
lose cells clump together and then spread to make a layer until it covers the embryo beneath the mesoderm before PS formation.
- then cells that are at the end start to produce descendants only at the posterior and these form tissue called the endoblast then start to push the original green hypoblast layer up
- then this endoblast come sin, the primitive streak starts to form
how did people start to identify what the role of the hypoblast and endoblast interaction and what did they find?
she looked for gene that were expressed in the hypoblast and not endoblast- cerberus was found which is an inhibitor of nodal (TGFbeta). the endoblast does not produce this
- she then wondered what happens when you take away the entire hypoblast
what happens when you remove the hypoblast from the embryo? what does this tell us?
you find that when you do this, you get many random primitive streaks- this suggests that the hypoblast is preventing PS formation and the endoblast has to remove the hypoblast to allow primitive streak formation
what do Vg1 and WNT8c activate?
nodal
what happens generally with the formation of the primitive streak?
cells at the PMZ express Vg1 and Wnt8C and then move upwards and as they start to move upwards the endoblast moves the inhibitory message (cerberus) away to allow those cells expressing nodal to then induce primtive streak formation as they move upwards via cell intercalation
what is troublesome about the fact a blastulae cut in 4 pieces can become 4 embryos?
the VG1 is only expressed in the PMZ, so other cells must start to express VG1 too
how can you start to look at the vg1 expression in a cut embryo?
take an embryo and cut in half, take one half and fix and do in situ for vg1.
- after 6 hours just about see vg1 expressed 50/50 on left or right in the corner on the light of the cut
- 12 hours later, nodal appears
then you get an axis starting
once you know that vg1 is upreg in one corner following cut of the embryo, how do you go about looking for the activitor?
you can do a a gene expression array to look for inhibitors on the vg1 non side and a vg1 activator on the relevant side
- the first way is to take the point opposite the PMZ in the normal embryo and do RNA seq
- you cut an embryo in half and straight away in situ for veg 1 in normal half. then you what 6 hours and cut two sections from either left or right corner. Then you do in situ and see on which side vg1 comes up- then you know where the left chunk or right chunk if the vg1 positive and which is negative- then you do RNA seq or microarray and compare gene expression
- you fin that 76 genes are up reg
what is a method of measuring RNA expression differences?
RNA seq
when is RNA seq not good?
when there could be lots of differences
what interesting gene was implicated in the expression of vg1 and why was this interesting?
- pitx2- it is involved in asymmetry- in identical twins- 25% of the time there are mirror images of each other - this suggests that a similar process involved in setting up left right sidedness is involved in this vg1 expression axis setting up
how did they test the role of pitx2?
- pitx2 in situ has same expression as veg1 over time and a bit earlier than veg1
- pitx2 upreg before veg1 in cut embryos
- do electroporation trick- introduce in cut embryo and put on the right corner and know that normal it is 50 50, if you KD pitx2 on the right-100% the axis forms on the left
how can you use bioinformatics to fine enhancers? then other techniques to check
- dont want to compare sequences between differ species these things change very quickly- can use a program to find non coding regions which might have similar functions because they have similar patterns for the vg1 gene and its homologues (?) - gives out 3 options find pitx2 binding sites within these similar regions of the vg1 gene- hten use chromatin immunoprecipitation and fine that the same regions come up - you then make enhancer construct using this trick using the implicated promoters - you find that one of them gives the same expression for vg1 in normal embryo
how does the AER maintain the cells beneath it in a mitotic state?
- it releases FGF8 which maintains the cells beneath it in a mitotic state. FGF8 released by the AER also induces the mesenchymal cells to releases FGF 10
how do the AER and the ZPA act in a positive feedback mechanism?
the AER releases FGF which stimulates the expression of fmn which activates Gremlin. gremlin then inhibits BMP which would normally inhibit FGF and the action of the AER. As the limb grows the FGF becomes further away and cassette ZPA to shift accordingly. The cells before become too far away from the FGF so dont express shh and then FGF also becomes inhibited in the preys mesenchyme so then they can differentiate
where do you inject the 3 MOs against the non-canonical pathway and where does the computer model inhibit the intercalation in the model?
in the posterior region of the epiblast
why has the chick hypoblast been said to be the homologue of the mouse AVE?
- similar gene expression- cerberus
- similar fate- become pushed anteriorly
- they both likely position the primitive streak
- dual role in protecting the prospective forebrain from caudilising influences of the organiser
in the chick, where does the definitive endoderm come from? how is this similar to the mouse?
it ingresses through the rostral tip of the primitive streak and becomes positioned under the anterior of the node, it then displaces the hypoblast. In the mouse the AME/ ADE migrates anteriorly and displaces the AVE (hypoblast) and is positioned under the anterior neural plate in the epiblast
what i the gastrula organiser of the mouse and the chick?
the node
what are the similarities in gene expression that the rostral hypoblast and mouse AVE share?
lim-1, otx2, Gsc, cerberus, hex and crescent
