Xenopus Flashcards
what are the two hemispheres of the zones embryo?
dark pigmented animal hemisphere
light pigmented vegetal hemisphere
what type of cleavage does the fertilised egg undergo?
holoblastic cleaveage
when does gastrulation begin in the xenons embryo?
after 10 hours
how can a fate map of the cells in the amphibian gastrulae be determined?
you can label cells and regions of cells in the vital dye and look at where and what tissues they end up forming in the tadpoles
what does the animal pole of /the amphibian gastrulae form
epidermis, nervous system (ecotderm)
what does the marginal zon/e of the amphibian embryo form
mesoderm
what does the vegetal pole of amphibian embryo form?
endoderm
what is formed above the dorsal lip of the amphibian embryo ?
dorsal tissues, notochord and nervous system
what is the exteneral process of gastrulation in the amphibian embryo?
- the blastopore lip extends around the entire circumferene of the amber before closing over the vegetal hemisphere- the blastopore closure marks the end of gastrulation. The near plate is just visible on the dorsal surface of the late gastrula
how do the germ layers interact with each other during/ amphibian gastrulation
the mesoderm and the endoderm invite into the blastocoel and migrate along the inner surface of the animal hemisphere. Involution is both initiated and more extensive on the dorsal side. A new cavity called the archenteron is formed which will form the lumen of the gut. The blastopore forms the anus.
at the end of gastrulation, what is the state of the nervous system?
the nervous system is a simple, flat, epithelial sheet on the dorsal side of the embryo called the neural pate . During neuralation the edges of this sheet elevate and move toward the dorsal midline, where they meet and fuse to form the nearl tube. This tube now lies beneath the doral epidermis
what does the endoderm form
- blood organ, liver, gut
what does the mesoderm form?
somite, muscle, cartilage , kidney
what were spemann’s seminal experiments and what did it show? (2)
- he separated each blastomere of a 2 cel newt embryo and found that they formed a normal half-sized larvae
- he then separated at a later time and used the dorsal lip as a marker to divide gastrulae into dorsal and ventral halves and found that the dorsal half formed a normal half-sized larva, while the ventral half produced a ball of epidermis and blood he called the belly piece- He suggests that this showed that the blastopore lip is required for regulative development
how can a specification map be made?
cut the egg into small fragments and culture them in vitro. how cells differentiate in vitro is know as specification
what can comparing the specification maps and fate maps of the tisues at the same stage of development- for example the gastrula, tell us about the patterning process?
- comparing the fat map and the specification map we can see that the nervous system, most of the somatic tissue, the heart and pronephros are not specified in the early gastrulae
what was the spemann experiment which looked at neural plate formation?
did an experiment to look at what points in development the near plate was specified. He replaced the ventral ectoderm (epidermis) with dorsal ectoderm (neural plate) and found that dorsal ectoderm always formed epidermis if transplanted at the beginning of gastrulation neural plate if transplanted at the end- he concluded that the nervous system is specified during gastrulation. Te innerness but the same principle was found for the epidermis when transplanted onto neural plate
how was the spumone organiser identified?
Mangold transplanted the dorsal blastopore li of an early gastrula to the ventral side of a similarly stages host. She found that a second dorsal axis was formed on the ventral side of the host in which the notochord was always formed by the transplant. However, the neural tube, somites and kidney were predominatly formed of host cells that would normally have formed ventral mesoderm (blood & mesothelium) and ventral ectoderm (epidermis). The transplant was able to respecify host ventral tissues to form a correctly proportioned, and patterned, second dorsal axis. Spemann called the dorsal blastopore lip the “Organiser”… it can change the fate of surrodunign cells
what is the gastrula organiser in the amphibian?
dorsal bastopore lip
what is the organiser in the fish?
shield
what is the organiser n the chick?
hensen’s node
what is the organiser in the mammal?
node
what did mangolds experiments suggest about the nature of the dorsal lip?
it was releasing inducing signals that dorsally the mesoderm and induce the mesoderm
what were the two theories about how the organiser was inducing the nervous system and how was the really method determined?
- there cold either be a planar route in which the signals from the dorsal lip spread upwards to the animal pole, forwards onto the mesoderm and downwards onto the vegetal pole
- the other idea was that there was a vertical signal from the underlying mesoderm that signalled to the overlying epidermis to induce neural tissue
- a way this was investigate was an experiment in which an traitors gastrulae was incubated in a high salt solution and found that the blastocoel collapsed. The mesoderm and endoderm ‘exogastulated’ creating an ectodermal that failed to form neural issue because it had no underlying neural tissue to signal to it- therefore it was vertical signalling
how can you check whether a tissue is a source of a inducing signal?
- remove it to see if the tissie being exposed still has the same fate
- or transplant and see if it can induce secondary tissue formation elsehwere- organiser
how did spumone and mangold initially try to identify what the neuralising factors from the underling mesoderm were, what were the problems with this approach?
- they took an animal cap which forms epideermis in vitro- its speciliastion is epidermis. they then added potential factors on to it and looked to see if it formed neural tissue- but the found that many chemical induced neural tissue
following the finding that many factors could induce epidermis to form neural tissue in vitro, what made the unspecifcity of the factor even more emphasised? (2)
- an experiment found that even dead organisers killed by eat or freezing, could induce neural tissue in the ventral ectoderm
- then an experiment found that even a grain of sand could induce neural tissue- used sox 3 was a neural tissue marker
what two experiments suggested that it was a stress response which triggered epidermis to become neural tissue ? what was the conclusion following this
- animal caps could differentiate as neural tissue in the absence of added factors, by altering the pH of the culture media- this shows that the cells has autonomous propensity to become neural - caps responses to mild dissociation by forming neural tissue- he ssuggested that animal caps contain repressor of neural differentiation that was blocked by the organiser
how did they go about finding organiser specific genes?
They investigated the molecular basis of the organizer by isolating mRNA from excised dorsal lips [at stage (st) 10.25 gastrula when the dorsal lips are capable of inducing a complete secondary axis. We then constructed a cDNA library from this mRNA, which is presumably enriched in molecules encoding proteins that provide positional information specific for the organizer, and screened for molecules that are likely to be involved in conferring organizer activity. They then used a labelled oligonucloetide probe of different protein as which they thought may be implicated such as those containing homeobox domain. This uncovered homeobox transcription factors sc h as goosceoic and encoding secreted proteins such as chorine.
how was chordin uncovered?
they isolated the RNA from dorsalised LiCl treated xenopus and form ventralised UV treated embryos. The mRNA was detected by their poly (A) tails with olig(dt) and was used this to synthesize cDNA single strands with reverse transcriptase 32p labelling. then these probes were hybridized with a dorsal lip cDNA library in order to detect those that were xpressed in LiCl in comparison to UV treated to identify dorsal expressed genes. They then looked for probes which were enriched in the dorsalised and not the ventralised- they found chordin- they then did in situ hybridisation and found it was expressed in the spemann organiser. Then they looked to see if it was expressed in the embryo where goosecoid had been injected which was known to result in a secondary axis by using whole mount in situ .
how was chordin showed to be able to mimic the action of the organiser? what does its injection fail to do?
injection of chordin mRNA into ventral blastomeres induces conjoined twin with neural tube, notochord and somites. But chords does not induce the most postal tissues including the forebrain and hindbrain
ow was noggin discovered? (in a different way to chordin)
- They isolated RNA from dorsalised gastrulae and then pools of RNA were injected into dorsalised (UV treated) embryos to identify any that rescue dorsal development.those pools that were selected were then split into sub pools etc until a single clone was found inject organiser-specific mRNA into ventralised embryos A gene encoding a novel secreted protein called noggin was identified using this assy and it was shown that it could also induce a second dorsal axis when the mRNS was injected into ventral blastomeres of normal embryos- although like cordon is could not induce the most rostral tissues.
that are the different expression levels of the organiser specific transcripts and how was this identified.
in situ hybridisation of chord, noggin, follistatin- d that chordin is the most abundant, noggin is less abundant. follistatin is the least abundant and weakest dorsalising agent
how have the three dorsalising agents been shown to induce neural tissue?
- the animal cap as placed in vitro and chordin follistatin and noggin were all applied. noggin has the greatest activity and follistatin has the weakest. - they all induce rostral character tissue such as forebrain
if chordin is the organiser signal, how would be expect it to act?
diffuse way from the organiser
hat was a key clue that chordin acted across the dorsal ventral axis?
Endogenous Chordin protein diffuses in Brachet’s cleft, a thin extracellular region that separates the ectoderm from the mesoderm and anterior endoderm. It is found on both dorsal and ventral sides of gastrulae, but at ~5 fold higher concentration on the dorsal side (beneath the neural plate).
what do the experiments showing that the addition of the 3 dorsalising demonstrate and why is this not enough?
they demonstrate that they are sufficient induce the nervous system but not whether they are necessary
how can you test whether the dorsaising signals are necessary to pattern the nervous system?
AMO are powerful tools for inhibiting gene function. They bind to complementary sequences in the target mRNA, either in 5’-UTR or at an exon-intron boundary. The former
blocks translation of the target mRNA and the latter splicing of the target mRNA (to produce a truncated protein) of the target mRNA. Since Xenopus laevis is tetraploid, and has four copies of the chordin (Chd) gene, two AMO were designed (one for each gene). Alone, each AMO reduced the amount of Chordin protein but had no effect on development. Together, they eliminated Chordin protein and embryos had greatly reduced heads. The notochord is missing and the ventral blood
island expanded. The phenotype was rescued by coinjecting Chordin mRNA that was not inhibited by the AMO.
once you have used AMO to reduce the expression of chordin, what is the final proof that you need?
- need to show it could be rescued- you inject mRNA for chordin that uses a different triplet code so that the AMO can’t target it- it reduces it
what was the key experiment which showed that chordin was the signal from the organiser?
Organisers were isolated from labelled AMO injected embryos and transplanted into the ventral side of host embryos. A Chordin AMO injected organisers did not induce a second dorsal axis in the host. Thus Chordin is essential for the dorsalising and neuralising activity of a transplanted organiser.
what makes xenopus leaves bad for genetics?
it is tetraploid
because laevis is tetraploid, what other xenopus can be used?
tropicalis
how was chordin shown to be the dominant actor in dorsal development?
Chordin (C), Noggin (N) and Follistatin (F) AMO were injected into embryos of Xenopus tropicalis. Only Chordin- MO, or combinations including Chordin-MO, had any effect on development, causing mild rostral defects. Injection of all three AMO caused loss of the nervous system (Sox2 and Sox3), notochord (Shh and Xnot) and myotome
(Myf5 and MyoD), and expansion of ventral tissues (not shown).
ow is SOG (fly) similar to chordin?
- shares 27% amino acid identity
- has 4 repeat of similar sequence- the same domain in dros appears in the same arrangement.
how was SOG linked to chordin?
At the same time two papers were published, one with the chordin sequence and the other with the SOG sequence - they realised they were similar
After it was found that SOG and chordin has similar sequence, how was SOG shown to have a similar function? and how was a dorsalising factor from the xenopus shown to act in a similar way in flies?
Injection of sog mRNA into ventral cleavage stage blastomeres induces a partial secondary axis, containing neural tissue and paraxial mesoderm but lacking a notochord and rostral tissues. SOG has similar biological activity to Chordin this was done by making an mRNA of the SOG gene and injecting it. Noggin is able to partially mimic SOG in fruit fly which carried a mutation of the SOG gene and it partially rescued the defects
what can’t you get to of a SOG secondary axis?
only partial because you dont get notochord- chordin generates a notochord
how did looking at the role of SOG in dorsal ventral patterning in the fruit fly, hep understand the role of chordin?
when you mutate SOG, you lose the neurogenic ectoderm and have an expansion of dorsal ectoderm. DPP has the opposite effect as SOG- mutants show an expansion of the neurogenic region and loss of the ventral tissues. In situ of both of these showed that dip is expressed ventrally and sog has an almost complimentary expression pattern. DPP specifies dorsal epidermis and SOG is required to prevent DPP from epidermalising the ventral neurogenic zone. only chordin of the 3 xenopus genes has a relevant homologue. we already knew how dros patterning works: dpp is the signal which regulates DV patterning. There are genes which modulate Dpp such as screw, tolloid, twisted gastrulation, crossveinless etc- mutations in all of these genes cause phenotypes that are weaker but similar to dpp
is the dpp SOG pairing found in many multicellular animals?
yes they do pretty much the same in thing in almost every mutli cellular animal
what is a trait of DPP and SOG?
both secreted proteins
how can you label protein diffusion?
label dpp gene with a GFP protein in frame as a translational translational reporter
at do you see when you label the endogeous Dpp and SOG protein gradients
dpp released from the dorsal and diffuses ventrally- opposite for the SOG
what is dpp a homologue of ?
BMPs
what experiment shows how similar BMPs are to dpp in the fly?
can take a fly dpp mutant and a human BMP4 and express it under the DPP promoter and this will reduce the fly
which BMPs are dpp very similar to?
BMP2 and BMP4
if you want to see whether two proteins are functionally equivalent across species, what 2 things must you do?
you must see if both can replace the other in mutants across the two species
list the DV proteins of the fly and their homologues
dpp= BMP
screw= BMP
SOG= chordin
tolloid= mmp
twisted gastrulation=BMP/ chrodin binding protein
crossveilness=BMP/ chrodin binding protein
what is the model of DV patterning in flies
- in the neurogenic ectoderm, SOG is produced, SOG diffuses towards the dorsal surface.
- BMPs form dimers- need two BMPs for it to work can form homodimers: dpp-dpp or screw-screw or they can be dpp-screw
- we know that homodimers can signal but are very weak- het is very strong - sog binds to het dimers preferentially. in the region where SOG is being released in the ventral region- it will bind all of the heterodimers and the dpp screws are diffusing from the dorsal towards SOG, as the get more ventral they meet more SOG and bind and SOG carried it in the opposite direction towards the dorsal side- facilitated diffusion. If doesn’t pick up the homodimers so these go ventral and signal in the neurogenic region.
- dpp and screw bind to SOG very efficiently if they also bind to TSG on the chordin-
- tolloid is made on the dorsal surface and doesn’tdiffuse very far - only see SOG when it gets tot he dorsal side and when it reaches here it cleaves SOG and causes the heterodimers to be released which activate strong signalling to generate more dorsal tissues.
- this explains why the knockout of SOG causes ventralisation- need the facilitated diffusion to carry the dpp and screw to the dorsal at high levels- they simply are spread across the embryo and switches off the nervous system- can’t have any het signalling of nervous system.
where do Bone morphogenetic proteins get their names from?
injected of BMP can convert connective tissue to bone
what super family are BMPs part of?
TGF-beta
what is the structure of BMPs?
- they have a cytosine rich region and upstream of that an RxxR region which is the cleavage sight
are BMPs highly conserved across the animal kingdom
yes very much so
what must BMPs form in order to function?
homodimers
what do BMP4 dimers bind to ?
specific type I and type I serine threonine kinase receptors both a once
what is the bMP4 signalling cascade?
they bind to type I or type II serine threonine kinase receptors, this stimulates RII to phosphorylate RI . they RI phosphorylates smad1 that forms a complex with smad 4. This complex enters the nucleus and acts as a cofactor to regulate transcription of target genes such as Vent1 and msx1.
what genes can the BMP4 signalling pathway activate
vent2 or msx1
hat generally happens to the embryo when BMP4 is over expressed?
it becomes ventralised
where is BMP4 expression lost from in the early gastrulae ?
it is lost from the organiser
where is BMP4 expression lost from in the last gastrulae?
neural plate- after being lost from the organiser
where is BMP4 expression restricted to?
the ventral and lateral regions of the embryo but not the organiser
where is BMP signalling strongest in the embryo and what shows this?
ventral portion of the embryo- tams labelling shows that smad1 has moved into the nucleus of the embryo opposite the dorsal lip
what happens when yo inject BMP4 into the entire embryo?
the ventral tissue expands at the expense of dorsal tissue- this is identical to the embryos lacking the organiser
how does the injected of BMP4 into the entire fertilised egg affect the expression of chordin ?
it is initially expressed but then turned off in the gastrulae
what is the affect of injecting AMO for BMP2, 4 or 7 into the fertilised egg?
has a mild dorsalising effect with the severity increased by injected pairs of BMO- dorsalising activity was the strongest following injection of all three MO, demonstrating overlapping function of these BMP.- you see this by using in situ to stain for markers of dorsal tissue such as moD for skeletal muscle, krox20 for transcription factor in the hindbrain, otx2 for the forebrain
in addition to using AMOs against BMPs, how else can you inhibit its functioning?
create a dominant-negative BMP receptor of the BMPR!- this is done by injection g ht RNA for the truncated protein. . Yo inject excessive amounts so that you can ensure most of the heterodimers of the RI and RII are formed between the double native truncated protein This works because RI is needed to phosphorylate RII- so without it you can’t have the signalling pathway
what does the fact that combined AMOs produces the most significant phenotype suggest about the three BMPs?
the have overlapping function
what is the naturally occurring DNBMPR allele called?
bambi
what happens when you inject the nBMPR1 receptor into the ventral side of the embryo and what does this suggest?
induces a secondary axis- just like chordin does - this suggests that inhibition of BMP4 results in neural tissue- this suggests that neural tissue is the default and that BMP signalling is required for ventral tissue
how was it fond that BMp4 induces epidermis?what does this suggest?
- they took an animal cap and allowed it to differentiate- it forms epidermis
- they applied nBMPRI to the nail cap in vitro and they get neural tube
- they induce dissociation of the animal cap cells and get neural tube
- when they apply BMP4 to dissociated animal cap cells they get epidermis
- this suggests that neural differentiation is the default pathway of ectoderm
after determining that neural tissue is the efualt, what is thought to be the general way in which neural tissue is patterned?
neural tissue is the default but BMP signalling suppresses this to induce epidermis- then you need chordin to suppress Bmp signalling to allow neural tissue to form
how are dorsalising factors thought to inhibit BMP?
- biochemical studies have shown that chordin and noggin can bind BMP2 and BMP4 with high affinity and follistatiin can bind bMP7. Bound BMP are unable to interact with their receptor and do not activate intracellular signalling
which BMPs do chorine nd noggin bind?
BMP2 and BMP4
what is the definition of a morphogen?
a secreted protein which can activate more than 2 different tissue types over a gradient concentrations
how was it tested whether BMP4 acts in a concentration dependent manner to induce different ventral tissues?
4 cell stage embryos were microinjected with either low levels of BMP4 or high levels of BMP4. The extend of venralisaton was determined by carrying gout in situ hybridisation for dorsal markers such as chordin and xmyf5 and ventral markers such as vent 1 and 2.
- Consistent with this, higher concentrations of BMP inhibitors (e.g. Noggin) have stronger dorsalising activity than lower concentrations.
This suggests that BMP4 might be a morphogen, specifying different fates at different concentrations.xvent1 and 2 expression spread around the entire circumference of the embryo with high BMp4 levels but this was less so with low levels. Chordin and xmy5 expression was reduced with low BMp4 but not completely gone- but was gone with high doses
what would Mpp4 need to do to be shown to act as a morphogen?
act in a concentration dependent manner to induce ventralisation
where is chordin expression low?
brachets cleft oppositethe organiser and dorsal lip
so if BMP acts over a gradient, how are the cells in the embryo thought to respond to different levels of BMP and how do they react?
the cells read off the different levels of BMP and express the different genes accordingly from high BMP to low BMP:
- sizzled, vent1 and vent 2, then lose sizzled, then lose vent 1 then gain xmyf5
in the xenopus, where is tolloid (xolloid-related) expressed?
localised to the ventral-lateral mesoderm of xenopus embryos where it is induced by BMP signalling.
what is the structure of tolled proteins?
proregion, metalloproteases, and a protein-protein interaction domain
how is BMP1 related to tolloid~?
it is a splice variant- lacks the last 2 cub domains
what induces tolloid expression?
BMP signalling
what happens to the blastomere when you inject tolloid into the dorsal tissue?
expands ventral and reduces dorsal tissues (SIMILAR TO CHORDIN AMO)
when these experiments are injecting to the embryo, when are they normally injected?
blastomere
what happens when you inject dn Tolloid mRNA into the ventral region?
produce a conjoined twin
what does the fact that inhibiting tolloid signalling at the ventral side induces a conjoined twin, and injecting tolloid into the dorsal side induces ventralisation, suggest about its role in DV patterning
thought to regulate BMP signalling
how does tolloid interact with chordin? how does this explain experiments relating t o its dorsal injection and injection of its dn.
in cleaves it chordin at two locations: after CR1 and between CR3 and 4- this increases the amount of BMP available to activate its receptors. This explains why injection into the dorsal side causes ventralisation- chordin is prevented from inhibiting BMP so BMP can act-. Furthermore it can’t disinhibit chordin wehen its DN is injected ventrally producing a dorsal phenotype
what is the role of Xlr on the chordin gradient?
it makes it- inhibits it ventrally b cleavage
why is chordin simply diffusing from the dorsal side to the ventral side not enough to produce a gradient?
- initially there will be a gradient but as the chordin keeps coming, it will eventualy even out- so you need a chordin inhibitor
what is sizzled similar to?
it has the WNT binding domain of frizzled receptor
what is sizzled induced by?
high concentrations of BMP
what happens when you inject xenopus embryos with sizzled mRNA?
they become dorsalised
what is sizzled?
a tolloid inhibitor
what happens when you inject a sizzled MO?
it ventralises the embryo by increasing BMP signalling which is shown by the fact in situ shows increase sizzled transcription (MO targets mRNA doesn’t stop transcription)
- they have less chordin proteins that controls
how does sizzled act?
in hibits cleavage of chordin by xolloid. It controls BMP signalling by regulating the availibility of chordin
how does AMo against tolloid and sizzled compare ?
Injection of Tolloid AMO (bmp1 & xlr) increases the Chordin signal detected in Brachet’s cleft, while injection of sizzled AMO decreases this signal. The activity of Tolloid regulates the Chordin gradient, and hence the BMP gradient, of Xenopus gastrulae.
what is crescent?
it has the same binding domain as frizzled for wnt and it binds and inhibits wnt proteins. its transcription is inhibited by BMP signalling so its mRNA is localised to the spumone organiser.
where is crescent expressed?
spemann organiser because it is inhibited by BMP signalling
what does RNAi injection of crescent cause?
cyclopia at the tadpole stage
what is the effect of crescent RNA injection into the embryo?
increases cordon expression
what is the effect of AMO injection against crescent? why is this surprising?
reduces chordin expression and expands sizzled. This is the similar activity of sizled- but they are at opposite ends of the embryo- very weird!
what is the result of co injection of AMO of sizzled and crescent and what does this show?
A phenotype similar to that obtained with Sizzled, even though they are expressed at opposite poles of the gastrula! Coinjection of crescent and sizzled AMO reduces chordin expression to greater extent than either crescent or sizzled alone. These results suggest that Crescent regulates BMP signalling
what are the actions of crescent thought to be?
crescent inhibits tolloid, thereby regulating the availability of chordin
how do sizzled and crescent inhibit tollioid activity on chordin?
- they bind to chordin and prevent tolloid from binding to cleave which means that BMP remains bound to chordin
what is twisted gastrulation?
it is a secreted protein with N-terminal BMP binding and a c-terminal chordin domain. It is expressed on the dorsal side of the late blastula (but not gastrula) then in the ventral mesoderm of the late gastrulaeIt is induced in ventral mesoderm by BMP signalling. Tsg increases the affinity of Chordin for BMP4, making a stronger BMP inhibitor. Whereas Tsg1 antagonises BMP signalling in the presence of Chordin, it may potentiate of BMP signalling in the absence of Chordin.
where is twisted gastrulation expressed?
Tsg1 (but not Tsg2) is expressed on the dorsal side of late blastulae (but not gastrulae) then in the ventral mesoderm of late gastrulae.
what induces twisted gastrulation expression?
BMP signalling
what is the role of TSG?
increases the affinity of chordin for BMP4, maaking it a strong BMP inhibitor
what is the result of injected TSG1 AMO into tropical embryo?
did not disrupt chd expression at stage 11, but reduced expression of myf5 (muscle) and expanded expression of sizzled (ventral mesoderm). At stage 25, sizzled expression is expanded and expression of eomesodermin (forebrain) reduced. Defects enhanced by coinjection of chd-AMO and rescued by coinjection of either chd mRNA or bmp(2/7)-AMOs.
what is the role of cross veinless?
a secreted protein with an N -terminal BMP binding and a c-terminal vWFD domain
what is the role of the vWFD domain of crossveiless?
binds to the ECM, restricting the movement to a few cell diameters
what does CV2 bind to ?
BMP, Tsg and chordin
what is the general role of CV2?
a context dependent antagonist/potentitor of BMP(antagonist in the presence of Chordin, potentiates in the absence of Chordin). Cv2 transcription is activated by BMP signalling on the ventral side of gastrulae.
what is the result of injecting CV2 mRNA into the ventral blastomeres?
induces a partial secondary axis
what is the role of AMO injection of CV2?
partially ventralises the embryo
what is the action of sizzled similar to?
chordin - it prevent tolloid from cleaving chordin so allows chordin to inhibit BMP on the ventral side
what is the role of crescent?
it inhibits tolloid so promotes the inhibition of BMP
what is the role of TSG?
Tsg1 (but not Tsg2) is expressed on the dorsal side of late blastulae (but not gastrulae) then in the ventral mesoderm of late gastrulae. It is induced in ventral mesoderm by BMP signalling. Tsg increases the affinity of Chordin for BMP4, making a stronger BMP inhibitor. Whereas Tsg1 antagonises BMP signalling in the presence of Chordin, it may potentiate of BMP signalling in the absence of Chordin.
how is Cv2 similar to chordin?
-Injection of full-length cv2 mRNA into ventral blastomeres induces a partial secondary axis, while AMO injection partially ventralises the embryo (increased cv2 and szl expression). This is similar to chd AMO, even though they are expressed on opposite sides of gastrulae! Coinjection of chordin AMO and cv2 AMO further expands cv2 and szl expression. Cv2 and Chordin cooperate to inhibit BMP signalling on the ventral side of the embryo.
how does TSG interact with the inhibition go BMP induced by Cv2 and chordin on the ventral side the embryo?
Tsg, Cv2 and Chd form a complex with high affinity, inhibitory, binding of BMP. Cv2 restricts movement of this complex by binding to the ECM and may also tether the complex to BMPR1. This complex is more efficiently cleaved by Tld than Chd alone, releasing Tsg, BMP and small Chd fragments, while the large Chd fragment may remain bound to Cv2. Tsg and BMP may remain bound, with the Tsg delivering BMP to the receptors.
what was the evidence for the link between tsg and cv2/chordin
Ventralisation by coinjected cv2 and chd AMOs is blocked by coinjection of tsg1 AMO, revealing a role for Tsg in potentiating BMP signalling. Dorsalisation by injection of Cv2 protein is also blocked by coinjected tsg1 AMO, but is enhanced by coinjection of Tsg protein. Suggests that Tsg is an essential regulator of Cv2 and Chordin function, perhaps through its interaction with these proteins in a BMP inhibitory complex.
what does the inhibition of all 3 BMPs result in and what does this suggest ?
Inhibition of BMP signalling dorsalises Xenopus embryos, with the strongest phenotype caused by inhibition of all 3 BMP (bmp2, bmp4 or bmp7). However, the embryos were not completely dorsalised! Are there additional ventralising signals?
what is another BMP-sigannling protein and where is it expressed?
ADMP- in the spemann organiser
what is the surprising phenotype of ADMP RNA injected into a fertilised egg?
Remarkably, it has the same phenotype as BMP4 in mRNA injection experiments; injected embryos are ventralised!
what is the result o ADMP MO injection/
weak dorsalising effect - the opposite to chordin MO injections
what is the result of connection both MO for ADMP and chordin? what does this suggest ?
gives normal development, indicating that a key role for both proteins is to regulate each other activity. ADMP has BMP-like ventralising activity that is inhibited by Chordin through direct binding.
what demonstrated that BMP signals from both the dorsal and ventral sides of the embryo are required for normal dorsal-ventral patterning?
Injection of of AMO for bmp2, bmp4, bmp7 and admp results in complete dorsalisation of the ectoderm, with expansion of neural markers (sox2, otx2, rax2a, krox20) and loss of epidermal marker (cytoK).- whereas loss of BMPS without ADMP doesn’t completely dorsalise
what demonstrated that the ventral marginal zone may also be a signalling centre that complements the better known dorsal organiser?
Grafts of both the dorsal and ventral marginal zone can partially rescue DV patterning in embryos injected admp and bmp AMO. The effect is long range, since host cells lacking endogenous BMP activity respond to BMP signals from the graft. Suggests that ventral marginal zone may also be a signalling centre that complements the better know dorsal centre (Spemann Organiser).
wha is the simplified see-saw model for DV patterning in the venous gastrula?
Dorsal and ventral inhibitors of Tolloid metalloproteases adjust the DV gradient of BMP signalling through cleavage of the BMP inhibitor Chordin. Transcriptional regulation is indicated with blue lines and direct protein-protein interactions with black lines.
what is the role of the cordon TSG, CV2 BMP complex generally?
the idea is that chordin bind BMP and tag makes it a tight binding then CV2 will bind and brin this compex near to the receptor- so anywhere other than at the ventral sid where tolloid i expressed- BMP will be inhibited- but because tolloid is in the ventral it will cleave the complex and allow the complexes that are already bound to a BMPR receptor- this will allow BMP tp released near to the receptor.
what is the thought to be the reason that embryos have these organisers at each end of the embryo?
Even though the embryos are half sized they form a nearly normal embryo in which all the tissues are suitably proportioned. Mathematical models have shown that this would not be possible with a simple model in which a gradient of Chordin creates a counter gradient of BMP (as in slide 12). What is required is a self regulating field with dorsal and ventral centres, each containing activators and inhibitors of BMP signalling. This is important because amphibian eggs are variable in size, both within and between species. The self regulating field allows for this size variation, creating a normal pattern whatever the size of the egg.
go to the model organism section from functional genetics and learn cards relevant from there
ffr
