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
