1
Q
when you are talking about the AP patterning processes, what are people generally talking about?
A
the patterning of the AP axis of the neural tube
2
Q
what is the difference between cell fate and specificaiton
A
- cells in a certain position that will give rise to a limb= cell fate. Fate- when you let development develop normally the fate that a cell will take- you measure cell fate by labelling them. specification is when a cell changes its gene expression so that it will form a certain tissue
3
Q
what is naive tissue?
A
has the capacity to respond to a broad variety of signals because it has not been specified - can respond to a variety of signals
4
Q
what is the anatomy of the early mouse embryo?
A
dpc= days post coetum- the days breed at midnight and you can check for plug
- the cells divide and after 16 cells division sat 3 days uou have a morula and then this becomes the blastocyst. This divides into two tissues- the trophoblast or the inner cell mass- the ICM gives rise to the embryo
- at implantation the inner cell mass divides into two types of cells- primitive endoderm (extraembryonic tissue mostly but some left over in the embryo) and the epiblast (embryo) then there is a blastocoel
- the primitive endoderm sinks into the cavity- surrounded by the endoderm at 5.5, at 6 the embryo there is no sign of what is A or P asymm is at 6.5 during gastrulation- the presence of the primitive streak which forms in the posterior- this streak elongates to the distal part and then forms the node - at the node the in the anterior part- there is the gut endoderm (definitive endoderm)- not the visceral endoderm which becomes replaced by the definitive endoderm derivatives
5
Q
to generally form anterior nervous system what needs to happen?
A
- WNT and BMP inhibition
6
Q
what is the anterior visceral endoderm?
A
it is the hypoblast in chicks
7
Q
what is the importance of the AVE?
A
it inhibits WNT signals at the anterior point in mouse development
8
Q
how do you find what the origin of the AVE?
A
- they did a labelling experiment- they labelled the the visceral endoderm and found that the distal visceral endoderm ended p in the AVE - they use DiI which gets attached to the membrane of the cell
9
Q
what is the axial mesoendoderm?
A
prechordal plate in the mouse
10
Q
from what is the AVE derived from?
A
distal visceral endoderm
11
Q
when they did in situ for HEx, what did they find?
A
that is was in the primitve endoderm- then the ditsal visceral endoderm then the DVE- opposite the primitive streak (maybe the AVE determines whether the primitive streak is going to be ) then they saw that it is expessed in the area of the posterior after the AVE has expressed the hex
12
Q
why did they think that the AVE was formed from the distal visceral endoderm initially?
A
because they had done in situ for hex and seen that the expression changes from the primitive endoderm and then in the ditsal visceral endoderm and then in the DVE.
13
Q
where is Hex expressed at 6.5?
A
where the axial mesoendoderm should be in the definitive endoderm
14
Q
what was seen about the Hex expression in the definitive endoderm in the node?
A
that the Hex expression is present in the AME and ADE and then this moves around with the node and replaces the AVE
15
Q
what is the role of the initaly expression in the AVE from cells derived from the distal visceral endoderm and the movement of cells expressing Hex coming from the node?
A
the cells from the distal visceral endoderm initiate the patterning done by the AVE for the patterning of the anterior tissue, then the cells from the node reconstitute these bcause normal dveelopment push the inital cells away and the cells of the node activate the same programme an dthe same inhibitors so that the area of the AVE will still maintain the same signalling mechanisms
16
Q
how can you label Hex expression?
A
use a reporter GFP hex consturct
17
Q
where does the AVE lie?
A
under the ectodderm that will give rise to the anterior neural plate
18
Q
in the region of the AVE that express Hex, what other egens are expressed in these cells?
A
genes such as WNT inhibitors
19
Q
how did they look at what the fate of the visceral endoderm was?
A
- initially, from labelling people thought that it become the extraembryonic endoderm
- but then they used GFP and RFP- they used RFP to label the visceral endoderm and green to label the definitive endoderm - they found that some visceral endoderm cells were expressed in the definitve endoderm- not just displaced completely to become the extraembryonic endoderm
20
Q
how can you see that some visceral endoderm forms some of the gut?
A
you can use GFP markers and in situ staining for markers of the visceral endoderm and you see it in al three sections of the gut
21
Q
what was the old and new model for the fate of VE?
A
during gastrulation the epiblast displaces the visceral enoderm by migratin anteriorly and then displaces it completely- bu the new model is that- the cells from the tip of primitive streak start to interacalate in the visceral embryo and displace it almost completely
22
Q
how did they go about finding the mouse organiser in the mouse?
A
- in the chick you transplant the hensen’s node then you get a secondary axis
- in the mouse this kind of test hadnt been done because the mouse is hard to transplant
23
Q
what was the classical view of the AP axis formation?
A
the node forms in the posterior- moves anterior and then this patterns the anterior neural tissue and the AP axis
24
Q
what was the experiment looking at the transplantation of the “organiser” in the mouse
A
at 7.5 days: she took mouse embryo and took piece of anterior endoderm and the node and transplanted individually - either the anterior ectoderm or the node into the lateral mesoderm- if the node was able to induce a full axis then you would see an axis - when you transplant the node you could never get anteriro neural folds
- you could get posterior ectoderm this show you need the AVE with the node
25
what happens when you remove the AVE? what did they tell us?
you can affect the patterning of the anterior neural plate- (Hesx1- expression in the anterior neural plate is reduced significantly) - this stells us that it might be important in the patterning of anterior neural plate
26
what genetic evidence was there for the AVEin particular being involved in the anterior neural plate?
Chimeras with Lim1-deficient AVE but normal ADE have the same brain defects as Lim1-/- embryos- they found that a lim1 mutant had no head- looked at the expression pattern of lim1 and found it have a pattern like Hex- in the AVE and ADE- you can generate chimeras to test in which of these this is needed- put wild type ES stem cells into a lim1 mutant - when you inject stem cells into the inner cell mass they normally do not colonies the primitive endoderm- this means that you will generate an AVE that is -/- but the defintive endoderm is +/+- this shoed there was still a truncation- showing the lim1 is required in the AVE
27
what were the general conclusions made from the AVE studies?
AP patterning is initiated prior to gastrulation by the AVE and later maintained by the node/ derivatives (AME, ADE)
• Several genes relevant for anterior patterning are expressed within the AVE (Hex, Hesx1, Lim1, Otx2, etc)
• Genetic evidence (i.e. knock-out and chimeric mice) supports the idea that AVE is required for anterior patterning
-
• AVE is not sufficient to bestow permanent anterior patterning:
For example, it cannot maintain expression of forebrain markers for several hours or induce a head.
• AVE is not a head organiser, is a signalling centre
28
is AVE a head organiser?
no it is a signalling centre- it needs the support from the node and the derivatives like prechordal plate etc
29
what does the AVE secrete?
It antagonises posteriorising signals: Wnt, BMP and nodal.
| • It secretes several secreted inhibitors such as Cerberus-1, DKK1, Lefty1.
30
what did the fact that lim1 is expressed in the AVE and is needed for head formation, before it is needed in the ADE?
this shows that the AVE is patterend before there is any neural induction and before the pirmitive streak - then the chimeric evidence and the KO gave evidence that the AVE is required prior to the ADE
31
how was it shown that the AVE was not an organiser? what was suggested due to this?
- transplantation of the AVE mostly could not form an entire axis alone- it can induce neural character but not for long enough - neural markers diminish after a while - so the AVE is a transient signalling component that is then maintained by the node- need to combine the AVE with the node to form a second axis
32
how did they show that an excess of WNT leads to loss of forbrain?
when you have a transgenic mouse expressing WNT8a the hesX1 marker becomes smaller- the cells in the anterior are respecified
33
so what is the general role of AVE in summary
- 5.5 the idtsal visceral endodderm cells express Hex and they are porgrammed to migrate to the anterior part of the mebryo and these cell express inhibitors of WNT and TGF-beta which are acting on the cells in the ectoderm
6. 5- primitive streak to formed in th eposterior and the then the axial mesoendoderm starts to migrate towards the AVE with the node
- the WNT inhibiton by the AVE and of BMP results in the expression of Hesx1 in the forebrain - the AVE protects them from being induced by posteriorising signals
34
what is the AVE equivalent in other vertebrates?
- see picture- very good! for the chick you flatten the mouse plane and in the xenopus the AVE is what is pushed via ingression - hex is expressed in the relative region under the anterior ectoderm and in the chick it is expressed in the hypoblast too
35
what happens if you dont have the AVE?
the tissue turns into the posterior neural tissue
36
what is nodal flow?
flow within the node during LR asymm development
37
what is the first characateristic of LR asymmetry?
nodal expression in the lateral plate mesoderm
38
what happens if you express nodal on the right in the lateral plate mesoderm?
get left isomerism- two lefts
39
what shows that nodal expression determines left patterning?
if you express nodal on the right of the node and in the lateral plate mesoderm you get left isomerism- two lefts
40
what is weird about the expression of nodal on the left of the node and the expression in the lateral plate mesoderm?
the laterl plate mesodemr is far away from the node!
41
how were cilia in the node first identified?
microscopy- you could see the cilia moving
42
how was the left nodal flow first seen?
if you put particles in the nodal flow you see that the is a leftward flow
43
how was the leftward movement of the cilia at the node shown to be important?
if you mutant the microtubules so no flow- you get two lefts
44
what are the two equivalents to the cilia in the note in vertebrates?
- the kupffer's vesicle in the zebrafihs, the gastrocoel roof plate in the xenopus
45
how do the cillia insitgate the leftward flow?
they tilt towards the posterior to stop just a vortex
46
is the leftward flow of the cilia the symmetry breaking step in mice?
no ! must be something which causes the leftward flow
47
what were the two models for the mechanism of nodal flow?
determinant molecules are sent leftwards or there is a mechanism stress on the left
48
why was the signalling molecule leftward movement shownto be worng?
didnt compute with viscosity etc
49
what is the idea of the mechanosensory mechanism?
the flow in the nodal pit activates asymmetric calcium signalling-
50
what happensif you interfere with the calcium signallinh?
interfere with nodal left expression
51
what is the calcium channel implicated with left right asymmtry?
PKD2
52
what is the evidence for Ca++ wokring via PDK2 in LR asymmetries
- Asymmetric Ca++ signalling has been observed in the left side of the node in wt embryos
• Elevation of Ca++ concentration and lateral propagation has been observed
• The Ca++ channel PKD2 has been implicated in LR asymmetry
• Conditions caused by abnormal cillia formation are associated with polycistic kidney disease and LR defects
• Planar cell polarity genes disrupt cillia movement and cause LR defects
53
what is the genetic pathway thought to be for LR in mice?
at the midlline tehre is nodal flow and there is nodal on the left which activates pitx2 and left2 which is requiref for left patterning
- and there is an inhibitory signal coming from the right (lrd or inv) which prevents the expression of lefty 2 - int LPM
54
what are vangl1 and vangl2?
- when you label the cilia you see that cilia are very disorganised in these mutants- they show that there is abnomral lefty and nodal expression by doing an in situ- there was no directional flow- did clacium imagining- calcium was osmetimes only on the left or right and sometimes completelt absent
55
when is the LR axis detemined?
around 7.5 very early
56
at what stage in the mouse embryo an epiblast?
E5.5
57
from where in the embryo are the ES cells taken from
the inner cell mouse- these are the only cells that can give rise to the three germ layers
58
how do the visceral endoderm cells becomes the definitive endoderm?
- they intercalate into the definitive endoderm
59
how can you test whether the ES cells can give rise to the three germ layers? (3)
put in culture and then use different tissue marker staining
- look at sclerotoma- take epiblast stem cells and inject into the mouse, they will form scelrotoma- use markers for the three tissue types and see that they give rise to lots
- generate chimeras- inject into chimeras- they very rarely populate the extra embryonic endoderm
60
how do you isolate pluripotent stem cels from the mouse?
- remove the inner cell mouse cells and put them into a dish and culture and you can add component to make a transgenic cell
61
what shows that ES cells are stem cells in vitro?
- they can self renew in vitro- but they don't do this naturally
62
what do the human stem cells look like?
- epiblast mouse SC not mouse ES
63
hat factors do mES cells require?
LIF and BMP (different to the factors that hES and mEpiES cells)
64
how can human ES cells and mouse epiblast ES cells similar?
- they depend on FGF and nodal/activin signalling
65
what do all stem cells suppress?
- Sox2, Oct4, Nanog
66
what are the three methods used to generate genetically modified mice?
- microinjection of DNA into fertilised mouse eggs (pronuclear injection)
- genetic manipulations of mouse ES cells and blastocyst morula injection
- genome editing
67
what 3 things can you do with transgenics via ES cell
- Ko and Ki
| - gene trap strategies
68
what 3 things can you do with microinjection into the pronucleus?
-• Gene regula)on studies to characterise promoters
• Over or mis-expression studies of na)ve or mutated proteins to
induce/rescue a phenotype
• Expression of markers (GFP, lacZ,...)
69
where is DNA injected to in the mouse fertilised egg?
- the male transgene
70
how do you carry out pronuclear injection?
- inject DNA consturct into the male pro nucleus and transfer into a host mother here it will implant and hopefully some of the descendents will express the transgene
71
what must you always add to the end of a DNA construct ?
pA tail to stabilise the mRNA
72
what is bad about the pronucleus gene expression?
- you dont know where it has inserted- disrupt
73
are transgenic chick embryos common?
no
74
how can you make sure that your reporter gene is expressed even when heterozgous?
use a very strong promoter
75
describe how you can do promoter analysis using the pronuclear injection?
for hex:
use transgenics:
- you take different regions of regulatory DNA for a gene with a reporter in the construct
- you inject and produce different embryos for different regions - you compare this to an in situ for the expression of the gene being reported
- you see that the reporter is only being expressed in certain regions and not in everywhere that the gene is being expressed
76
how can you produce entire GFP animals?
- inject a transgene that contains a b-actin promoter driving expression of GFP
77
what is the process of gene targeting in ES cells?
- the process is based in homologous recombination
- it allows exogenous DNA to be directed specifically to a region of the chromosome, but happens at a very low frequency.
- you create a construct that has 5' and 3' homologous arms.
- within the construct there is a positive selection marker such as neomycin which confers resistance to the cells that it is inserted into. This allows the cells to be grown in cuture and the successful ones to be identified when grown on neomycin.
- if you inject the targetting vector ti the morula, then recombination will occur
- then you place the ES cells into a blastocyst and transfer into a recipient mother- the offspring will be chimeric of the surrogate mouse will be a chimera- so only some of the offspring will be heterozygous
- you then make a homozygous
(you use two different coloured mice so that ou can see the chimeric mouse coat- over the gonads)
78
why would you use a KI?
- to test the phenotype of a certain allele
| - if this allele gives the same phenotype as a KO (when homozygous) then you know that this mutation gives a null allele
79
how can you insert the DNA into the ES cell?
- electroporation
80
what is the offspring gender that is isolated from the surrogate ES cell mother?
males
81
how long do you have to wait for a mouse to have a blastocyst after mating?
3.5 days
82
what is the process of injecting the ES cells into a blastocyst?
- microinjection
83
with a Knock out what do you insert?
just the resistance gene
84
how can you use KI to look at a disease model of a person, in a mouse?
- you find a mutation in the patient- generate a KI of this mutation for the gene -
then you compare the phenotypes in the mouse and the human - look at whether the mutation is - work out how it is interacting with the process
85
why would you use a CRe/LoxP method?
can control where and when the gene is being knocked out or having an affect
86
how can the position of the loxp/ FRT sites determine the outcome?
- in same direction- removed
- in opposite direciton- inverse
- if you have each on a different site then you can get translocation
87
why is Lox generally used more then FRT?
- yeasr function at 10 degrees but bacteria- so FRT less efficient in mammals
88
what drives the loxp recombination?
Cre tissues specific of heat shock specific promoter
89
how do you get a floxed allele into the mouse?
- create a DNA construct that contains the gene of interest surrounded by the loxp sits- then recombine in at the given position
90
are there catalogues for mice cre expression ?
yes
91
what is normal fate mapping and what are the drawbacks?
- you use a dye such as DiI and then label the fate of the cells but this can become diluted mice do not for as fast as other model ogranisms
92
what locus is used for genetic fate mapping and why is this a good fate map?
- it is expressed in every cell and in every stage of development or adulthood
- its KO gives no phenotype
93
what is the rprocess of genetic fate mapping?
- use HR to insert a consturct 'Sa- loxp- PGK-neo- tPa (stop transcription) loxp-ATG and reporter
then you cross with a mouse expressing Cre in certain cells
- the descendents of these cells will continue o express the reporter even if then dont express cre anymore - you cn trace the descendants of a particular cell that you labelled
94
how can you use genetic fate mapping ?
- you can identify an area of the embryo that expresses a certain gene and then use genetic mapping to follow these tissues
95
how can you carry out genetic cel-lineage tracing using Cre and tamoxifen?
- you can create a CRE that is fused toa estrogen receptor that is kept outside of the nucleus and can only be nuclearised when activate by tamoxifen
- you have a mouse that expresses tis construct
- then you cross with a mous ethat you have HR'd a floxed stop into the ROSA26 region downstream of a start codon but upstream of a GFP with a slice acceptor- when you add tmx the cre will enter the cells and cause the transcrpipton of GFP by removing the stop codon= temporal and spatially precise- if using an area without a specific spatial gene expressed or something
96
explain how the reconstitution of the crypts can be shown by using the Cre lox system?
- you carry out genetic tracing ut you cant use a cre for the entire gut via a gut promoter because you would have labeled the entire gut-
you want to have another level of control- have a cre that is expressed in the certain tissue and have temporal control- you can use this by expressing a tissue specific inducible Cre then you use he estrogen receptor fusion protein that is driven in a cetrain tissue - in ject tamoxifen into the gut when you know that the tissus specific gene is only expresses in a certain tissue (the lgr5 which will drive expression in the crypt stem cells). Then you can see that after a while the gut has been reconstituted by stem cells in the crypt because only the new cells will express the marker
97
how can cre loxp be used to kill cells at a given time?
- use the cre loxp system with the maxofen fusion cre and have a floxed stop downstream of promoter in the ROSA26 upstrem of a DTA with a spice accapeotr
98
how can you use cre to look at cancer mutants? when can this be used for?
use a floxed stop upstream of mutant cancer gene in a certain tissue in a tissue-inducible manner- cre + tmx
-kill stem cells and see if they are important in ageing or other processes
99
when do you need to add a 5'UTR and 3'UTR and when do you not?
when you are doing construct injection because dont know where it will insert but not in homologus recomb because where you are inserting will already have it
100
The expression of which genes mark the anterior neural plate?
Hesx1 and six3
101
what does AME stand for
anterior axial mesendoderm
102
what will the AME give rise to?
anterior definitive endoderm, prechordal plate, notochordal plate
103
where does the AME derive from?
the gastrula organiser and the node- from the tip of the PS and subsequently from the node, a population of cells expressing Cer1 and Hex move anteriorly within the VE to underlie the rostal neural epithelium.
104
how do the ADE cells migrate and interact with the VE cells? what does this result in
the encoderm cells from the prechordal plate and notochord plate initially move within the existing layer of VE and not underneath, therefore they intercalate with VE cells in their migration. - this results in some endoderm cells becoming trapped and contribute to the endodermal lining of the adult gut.
105
how have the inducing activities of the anterior AME been shown?
explant xpeiments have sown the capacity of these cells to induce and or maintain anterior character in the vert embryoo.
106
what does ablation of the AME result in?
forebrain abnormalities in the chick ADE results in forbrain abnormalties
107
what is the mouse equivalent of the AME?
the ADE, it epands under the anterior primitive streak and displaces the hypoblast
108
what does the ADE in the chick express?
hex
109
what does the loss of hex in the chick ADE result in?
loss of hesx1 and six3 in the anterior forebrain
110
what is the role of the AME in the mouse and what is it similar to?
it secretes inhibitors of BMP/TGFbeta and WNT pathways- protecting the anterior neural plate from posteriorising signals- it expressed dkk1 crew and nog and chordin
111
what factor was shown to be an important component of patterning in the anterior neural border how was this shown in mouse and chick?
in mice- removal of the anterior neural border reduced FGF8 signalling
- removal of the anterior definitive endoderm in chick results in decrease in FGF8 signalling in the anterior neural border
112
what is the AME important for in terms of establishing signalling centres within the neural plate?
establishing neural signalling centres in the anterior neural plate which are needed for forebrain patterning
113
summarise the general role of the AVE and the AME in AP patterning
the AVE arises from the distal visceral endoderm and expresses hex which is require for the induction of anterior neural character. The AVE also secretes BMP/TGF-beta and WNT inhibitors (cerberus, dkk) which ensures that the overlying epiblast becomes anterior neural plate and it protected from caudalising signals, this acts in conjunction with the gastrulata organiser. At stage 7 however, the AVE begins to decrease in size as the ADE/AME cells intercalate through the visceral endoderm. These cells also secrete BMP/TGF-beta inhibitors such as cerberus, dkk-1, noggin, chordin) which replace the AVE in protecting the overlying epiblast from caudalisaing signals and ensuring the expression of hesx1 and six3.
The AME also promotes the expression of the anterior neural border via FGF8 expression within the anterior neural plate which acts as its own anterior organiser
114
at what stage in the mouse does the AVE essentially get replaced by the ADE/AME?
7-7.5 dpc
115
what was the evidence in the mouse that suggested that the gastrula organiser (node) is not sufficient to induce anterior neural fate and that another organiser must be needed?
- the fact that if you transplant the nod you dont get anterior neural fate but if you do it with AVE then you do
116
what is the evidence that the mouse AVE is the homologue of the hypoblast?
id you transplant the rabbit AVE into the chick then you get secondary axis with anterior neural fate
117
explain how it can be dmeonstrated and where the AVE equivalent is in the xenopus
Although early grafts in Xenopus induce the formation of a complete secondary axis including the head later grafts only induce trunk structures. This has been interpreted as meaning that separate signalling pathways organise the head and the trunk. Overexpression of genes expressed in the organiser to see if this can induce trunk or head formation have been used to try to identify these pathways. Noggin and chordin (BMP inhibitors) induce trunk duplications, while nodal related genes induce complete axes and another gene cerebus induces only head formation. Cerebus is in fact not expressed in the node proper but just anterior to it.
118
in the mouse and the chick where do the definitve endoderm come from?
ingress through the anterior of the node and then move ahead of the node
119
what is the process of genetic lineage tracing ?
using cre with tmx to induce GFP labelling at a certain time so se what certain tissues from a certain point give rise to
Mechanisms o f Development
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