Chick-neuralation

Question 1

how did paddington go about looking for a neural ograniser? what did he find

Answer 1

• he took different parts of the embryo and saw whether he got the formation of a second neural plate- he eventually transplanted hensen’s node and got a second nervous system

Question 2

what was a default model?

Answer 2

that nervous system is formed from the inhibition of BMP which inhibits neural its formation- the organiser is a source of inhibitors of BMP = chordin noggin follistatin

Question 3

how do you find that BMP inhibition is not sufficient to stop neural fate- in the xenopus- when do you get an expansion of the neural plate, why is this?

Answer 3

• instead of taking the entire animal cap and instead target one cell whose fateis normally to contribute ti epidermis of the bely- if you inject a dn BMP inhibit or smad 6- you dont get expression of a neural marker - but if you target another cell then you do get BMP- this is because BMP inhibition can only affect cells at the border of the neural plate

Question 4

how was BMP inhibition shown be insufficient in the chick?

Answer 4

if you hit the same competent region of the embryo with any BMP inhibitor- noggin, chordin, dnBMP , smad 6- all at the same time- you do not get a a second neural axis

Question 5

what does the fact that BMP inhibition is not sufficient to induce a second neural axis tell us?

Answer 5

• neural induction is a multistep process

Question 6

how can you investigate how long cells need to see the organiser in order to have a second axis induced?

Answer 6

• you add the organiser, leave it for different amounts of time before you remove it, then remove and grow the organism

Question 7

how long doe the tissue need to be exposed to the organiser in order to express sox3 and then sox2?

Answer 7

sox 3 after 3 hours and it is transient. at 5 hours BMP inhibitors stabilise sox3 but do not induce sox2. and then sox2 after 12 hours

Question 8

how does removing the node at different times after transplantation reveal that there is a multistep process involved in neural induction?

Answer 8

• sox3 is induced first and then sox 3 a while after- also it takes a long time

Question 9

how can you ask at what time point in node transplantation does BMP inhibition prolong sox3 expression after transplant node removal? what does this show?

Answer 9

you add and remove node at different time form quail to a chick, then add BMP inhibitors when you remove the node - you add the inhibitors of BMP by inserting cells that have been injected with a chordin DNA construct. This takes 5 hours, for the cells to become chordin sensitive

Question 10

how can you go about finding out what it is that determines whether a cell become chordin sensitive after 5 hours of node exposure?

Answer 10

you want to see what is different in the transcription levels of cells that have seen the organiser for 5 hours and those that havent- you can carry out a cDNA differential screen, or you can just do RNA seq, or a microarray

Question 11

what gene did they find are induced by he 5 hours exposure to the hensen’s node?

Answer 11

• ERNI
• churchill
• many of the genes that are regulated in the early period are regulated by FGF8 not BMP inhibition

Question 12

how can you predict the structure of a protein?

Answer 12

can look at the sequence on a database and blast it

Question 13

how do you find out where erni is expressed in the neural plate?

Answer 13

do in situ on normal embryo- find that it is expressed broadly at the stag4- very early and then is basically gone by stage 7

Question 14

how can you find out what induces ERNI? what did they find ?

Answer 14

instead of an organiser transplant you can test many different beads soaked in different factors and transplant into the embryo- which one can induce erni? use in situ to view it. you find that FGF8 is a strong inducer

Question 15

how can you test whether FGF8 is really stimulating the upreg of ERNI via the node transplant? what do you find?

Answer 15

• you transplant the node in the presence of an FGF inhibitor (chemical that blocks phos of the FGF R or an artificial version of the R which will out compete the receptor)
• find that you get no ERNI induction and no sox3

Question 16

how do you test whether FGF is needed for the late induction of sox2

Answer 16

if you take the embryos and grow them longer that have been inhibited for FGF while leaving the node there, you find they dont express sox2 either- this shows that FGF is required for the late markers too!

Question 17

how and why would you look for when FGF, sox3 and ERNI are all expressed in the same place in a wild type embryo? what do you find?

Answer 17

you would do this to find where in primitive streak formation does this induction naturally take place- what stage in development is it important for

• you would do in situ for then at different stages in development
• you find that FGF is expressed very early before gastrulation in the hypoblast which induces ERNI above it before gastrulation

Question 18

what linked ERNI to binding dimerisation?

Answer 18

has a coil domain

Question 19

how can you test whether ERNI dimerises?

Answer 19

you can use a mammalian 2-hybrid system- in which you have ERNI expressed with a gal4BD downstream of CMV and a another gene downstream of CMV and VP16 and then co transfect into a mammal cell?? which expresses GFP downstream of UAS site- when ERNI was used at ‘prey’ the GFP was transcribed!

Question 20

how can you make a dn of ERNI?

Answer 20

• you know that it makes dimers so you can make a truncated form of ERNI that just expresses the coil and then electroporate into he embryo so that it outcompetes the normal ERNI and stops it was doing what it wants to do
• or you can mutant the phosphorylating structure of ERNI and then electroporate this DNA

Question 21

how can you mark where you have electroporated?

Answer 21

put a GFP in to consturct

Question 22

what do you find when you electroporate in a DNA construct of dnERNI?

Answer 22

you see that there is a line of cells where you have electroporate that express sox2- this shows that inhibition of ERNI can induce sox2 in the gastrula tissue

Question 23

what does the fact that ERNI dn induces sox2 expression suggest

Answer 23

that there may be something endogenous that is inhibiting ERNI

Question 24

how can you look for proteins that bind to ERNI\? what did they find?

Answer 24

you do a cytotrap 2-hybrid screen and use a library of DNA from the cell where ERNI is being expressed at the time and you find BERT which looks like the dn of ERNI

Question 25

how can you test that it is BERT that is inducing the inhibition of ERNI and therefore inducing sox2 expression in the tissue ? what did they find

Answer 25

• you can do electroporation just like you did with ERNI and see if you get the same effect- you do ! you can also do in situ and see it is compliments ERNI expression: upreg when ERNI is downreg

Question 26

if you are looking for proteins that are involved in the same process of a protein you have found, how can you check that a candidate is suitable?

Answer 26

• do in situ to see if they have the same of comlimpentary domains, you can see if it is induced by the same protein that induces the other protein, you can see if its mis expression has the same phenotype

Question 27

what is bi-molecular fluorescence complementation?

Answer 27

you have two molecules that emit a optical light when two molecules are close to each other- you attack either C-terminus and the N terminus and fuse them to your two proteins of interest and you put the two proteins in culture in a cell - and you look for fluoresce- this shows the proteins interact with each other

Question 28

when would you use BiFCo?

Answer 28

to see if ERNI/ BERNI/gemini all bind to each other

Question 29

how can you measure if one of the three proteins binds preferentially to another?

Answer 29

you use BiFCo and add the two fluo proteins in with one that isn’t fluo and see if you get light or not when you add different levels of the non flu- if you need to put a little in them you have preferential binding to not labelled one but if you have to put a lot in then there is not preferential

Question 30

what is the transcriptional repressor which binds to ERNI?

Answer 30

HP1gamma

Question 31

what clues did we have on what was activating sox2? (3)

Answer 31

we know that the N2 domain controls sox expression in the neural plate - now that whatever is the binding partner must bind to this

• know that BRM can bind to N2 and activate sox2 expression
• geminin binds to BRM

Question 32

what is the general way in which sox2 is turned on in the neural tissue

Answer 32

• in the basal state BRM binds to HP1alpha at the N2 enhancer domain
• in early pre-neural state: FGF induces expressed of ERNI then sox3 and phosphorylates ERNIand then later gemini. then gemini and ERNI bind to each other, then geminin out competes HB1 alpha for binding to BRM and displaces it forming an inhibitor construct of GEM, ERNI, HP1gamma (inhibits sox 2)
• then just before the point go sox2 expression, something turns on BERT and is a strong out competing inhibitor or ERNi and GEM and turns on sox2 expression but releasing ERNi from the construct and removes HP1gamma

Question 33

what is the point of this complex sox2 expression being so complicated?

Answer 33

it is a timing process- all of these things have to be expressed in order for sox 2 to be turned out- ensures sox2 is turned on at the right time - role of ERNI is to delay the induction of sox2 while gastrulation takes place

Question 34

what is churchill?

Answer 34

in the future neural plate, stays there- similar to sox2, it is inducible by fgf but slower than the others- needs 4-5 hours of node t one transplnated

Question 35

what is brachyury a marker for and what is its role normaly?

Answer 35

mesoderm - it induces the ingression of cells through to primitive streak to form mesoderm

Question 36

what is the interaction between brachyury and churchill?

Answer 36

brachyury is blocked in the cells that produce neural plate and it is thought that churchill would do this

Question 37

what is churchill and what does this mean about its interaction with brachyury?

Answer 37

a transcriptional activator 0 it activates something else that then inhibits brachyury

Question 38

once they thought that sip-1 might be how churchill it inhibiting brachyury, how did they test this?

Answer 38

they did in situ and saw the expression pattern was alms identical to churchill

Question 39

how did they investigate if sip-1 was a target of churchill?

Answer 39

they looked at the sequence to which the churchill protein will bind most strongly - they find a sequence and then ask whether there is this sequence in the noncoding regions of the sip1 gene - there are at least 12 binding sites for churchill

Question 40

wat does churchill inhibit in terms of cells in the primitive streak?

Answer 40

the ingression of cells through the primitive streak by inhibiting brahcyury - stops cells form making mesoderm and make sure they stay outside

Question 41

how do you investigate whether the expression of churchill is required to stop ingression of cells?

Answer 41

• target cells that are right next to primitive streak in cells that will not ingress and then inhibit churchill via putting a MO into the cells- do these cells now ingress
• do a control morpholino labelled with fluo and target cells next tot neural plat then you see that they are all in the neural plate and have not ingressed- you then target the same location with the churchill MO
• you see that the cells have ingressed into the mesoderm when you cut through it

Question 42

what is the general model of or

Answer 42

• before gastrulation there is a hypoblast which produces FGF
• FGF induces ERNI and sox 3- the cells are not yet committed
• then development goes on then FGF induces nodal in the bottom cells but the activity of nodal is blocked by cerberus and then the nodal activity is freed and the primitive streak is induced which then requires brachyury, then the cells ingress
• meanwhile the hypoblast coninutes to express FGF
  once the streak is full length, the cells receiving FGF for a long time turns on churchill which turns on sip1 which blocks brachury which stops the cells form going in to form mesoderm and then sets the cells which will form neural tissue which will then go on to form neural tissue
• FGF ensures that it makes mesoderm in the early gastrula and not late gastrula by induced nodal and also ERNI (which blocks sox 2 expression) then after a while the churchill is turn on and stops brahcyury and then bert comes on and allows sox 2 to be expressed

Question 43

if you do RNA seq and you get thousands of genes, how can you try to narrow this down?

Answer 43

you can look for transcriptions factors- brings the number down a lot

• take the TFS and did expression anywise via in situ for all of them and found that they fall into different classes- some are ERNI like- early, some are sox3- in prim streak and remain, some are sox1 so expressed in early neural plate an some are sox-1 like at 12 hours onwards- they all seemed to fit into these 4 categories and came on at the same time
• then used nanostring: make probes for genes you’re interested in that have barcodes and then hybridised RNA to the probe and reveal which of the barcodes are present

Question 44

when are ENRI and sox 3 expressed?

Answer 44

before gastrulation

Question 45

when is FGF8 expressed?

Answer 45

in the underlying hypoblast before gastrulation

Question 46

ow did they show that the hypoblast can induce ERNi and sox3?

Answer 46

when transplanted into the marginal zone it can induce the expression of Henri and sox 3

Question 47

where is gemini expressed?

Answer 47

in the chick neural plate

Question 48

how did they show that FGF8 can induce gemini?

Answer 48

they transplanted it into he marginal zone and they did in situ and they found that ut was unregulated when fgf8 beads where putt there

Question 49

what did they show was the link between gemini and sox2?

Answer 49

its missexpression could give rise to sox 2

Question 50

where is churchill expressed

Answer 50

at stage 4 in the lateral regions of the epiblast and then in the neural tube- it is induced by the node (FGF8)

Question 51

what is the role of FGF initially before `bert is turned on?

Answer 51

to activate mesoderm with nodal