Cell fate determination - Localised determinants

Question 1

what are the events that lead to the formation of the xenopus embryonic primary nervous systems?

Answer 1

primary - allows embryo to swim after 2/3 days of development

some cells become part of 100s of neurons in 3 stripes either side of midline (motor, inter then sensory neurons) seen in the in situ hybridisation dorsal view of neural stage embryo
others don’t
what are the prerequisite events to get the pattern of neurons?
neuronal cell fate is determined by
spotty pattern forms how?

Question 2

how do cells take on a particular cell fate

which play a role in primary neutron formation

how many cell fates need to be determined before that of a primary neutron from the egg stage

Answer 2

mechanisms = 
1. localised determinants
cell signalling:
2 induction (+ morphogens)
3. lateral inhibition

4.

Question 3

maternal localised determinants

Answer 3

hypothesis:
1. determinant is synthesised in the oocyte = cell will become egg while still in the mother

2. in early development will become localised to a specific region in early embryo
3. subsequently only those cells that inherit that cytoplasm that contains determinant that will have specific fate that depends on the determinant

Question 4

how to determine whether a cells fate is determined by a maternal localised determinant

Answer 4

1. what is being localised in cytoplasm? either a RNA or protein ready made thats been localised
2. when is the determinant localised? either within the single cell oocyte or single cell embryo before it starts dividing or later
3. how is it localised?
   what forces are pushing cytoplasm into different regions counting this determinant
4. is the determinant necessary (loss of function) if you don’t have it you don’t get the cell fate
   and gain of function - is the determinant sufficient to impose a cell fate on what wouldn’t normally take that fate
5. how is the activation of the determinant regulated? if localised in a specific region of a single cell egg - might wait until cell divisions have occurred so that it only affects 1/16 of cells after a few rounds of divisions - how will we determine when the determinants has been activated
6. to regulate cell fate we have to affect gene expression
   any determinant must be a transcription factor - Tfs will effect gene expression,
   altered gene expression = particular cell fate

is it a transcription factor and what are the target genes that this determinant is activating/repressing

Question 5

is beta catenin a maternal localised determinant?

the role of dishevelled beta catenin and the organiser in the setting up of the dorsal ventral axis in xenopus

Answer 5

egg about to be fertilised:
dishevelled proteins are localised at the vegetal pole (bottoms side of embryo)
upon fertilisation dishevelled proteins relocate to one side defining the future dorsal side of the embryo
moving there is led by 2 processes
1. (slow) cortical rotation - outer part of egg cytoplasm rotates relative to central core of oocyte
as dishevelled protein is in the outer cortical region of the cytoplasm it gets moved around to the dorsal side
2.@the same time dishevelled combined with kinesin protein and GBP
when xenopus egg is fertilisation occurs there is a reorganisation of the microtubules; start of point of fertilisation and extend to the other side - the antipodes to the side of fertilisation
kinesin is a transport protein using ATP to move GBP and and DSH to one side = future dorsal side
= an accumulation of disheveled(component of wnt signalling) and GBP
when wnt signalling occurs dishevelled is activated = inhibits gsk3 - kinase which causes a break down of beta catenin
when dishevelled is present gsk3 is inhibited = beta catenin is stable on the dorsal side
antibody stain for beta catenin = since stabilised can enter nucleus where it has some effect whereas on ventral side beta catenin gets phosphorylated but gsk3 = broken down

Question 6

how does dishevelled protein stabilise beta catenin?
what does stabile beta catenin do?
what is the localised determinant?

Answer 6

dishevelled is a component of wnt pathway
wnt binds to the frizzled protein receptor activates dishevelled
absence of wet gsk3 interacts with beta catenin = degraded

when dishevelled is activated by wnt: gsk3 is inhibited and beta catenin is stabilised and accumulated
can enter nucleus to interact with TCF = forms active transcription factor

in early embryo,
localised dishevelled that starts at the vegetal pole
when it becomes localised and released from the kinase, it becomes active and inhibits the activity of gsk3
beta catenin is no longer metabolised and can enter the nucleus

dishevelled and GBP are localised but aren’t transcription factors and at the end beta catenin is localised and a TF because:

in the absence of beta catenin on the ventral side the tcf3 proteins repress the activation of the CM1
on dorsal side the combination of beta catenin with tcf3 protein = active transcription factor transcribes the siamois gene = siamois protein = activates gooscoidgene which can duplicate the axis found on dorsal side of embryo
if embryo is manipulated such that siamois is expressed on the ventral side and dorsal side = embryos with 2 main axes

Question 7

is beta catenin a determinant localised maternal

Answer 7

in the cytoplasm beta catenin is being localised and then moves into the nucleus

it is localised following fertilisation and up until the blastula stages

it is localised by the localisation of disheveled which inhibits gsk3 resulting in stabilised beta catenin.

activation of the determinant is regulated via the localisation of dishevelled moving onto the future dorsal side, allowing the accumulation of beta catenin = enter nucleus

transcription factor that works in combo with tif target gene is siamois gene critical for dorsal ventral patterning

Question 8

can the siamois protein be considered a localised maternal determinant ?

Answer 8

transcription facor
activates gooscoid
only works on dorsal side of embryo as only transcribed on dorsal side

not a maternal determinant
its being transcribed zygotically

its not localised in the egg but in the embryo following transcription whereas beta catenin protein and dishevelled are both present in the egg

Question 9

Ascidians have the same structure as an embryo as a tadpole or a mouse or human

they’re non vertebrate chordates

have a neural tube and notochord and bilateral somites and muscle blocks

one of first to have genome sequenced

undergo a metamorphosis

Answer 9

4 cells if grown can grow 4 different embryos with mostly different cell types but don’t form complete embryos like a sea urchin can
one contains muscle and others little or no muscle

@8 cell stage lower case = head
upper case = tail
B4`1 gives rise predominantly to muscle 
mesoderm endoderm
A4.1 = endoderm notochord
B4.2 ectoderm
A4.2 = ectoderm

Question 10

macho -1 s a maternal localised determinant in the ascidians

Answer 10

one pair of blastomeres (posterior vegetal; B4.1) the 8-cell embryo is capable of producing ta muscle tissue (Whittaker 1982). These cells contain the yellow crescent cytoplasm. When yellow cres­ cent cytoplasm is translerred from the B4.1 (muscle-form­ ing) blastomere to the b4.2 (ectoderm-formin g) blastomere of an S-cell tunicate embryo, the ectoderm-forming blastomere generates muscle cells as well as its normal ectoderm

cytoplasm from the yellow crescent area of the fer l­ ized egg can cause the a4.2 blastomere to express muscle­ specific proteins

certain determinants present in the egg cytoplasm cause the fonnation of certain tissues. These morphogenetic determinants appear to work by selective­ ly activating or inactivating specific genes. The determina­ tion of the blastomeres and the activation of certa genes are controlled by the spatial localization of the morpho-
enetic determinants within the egg cytoplasm.

Using RNA hybridization techniques, Nishida and Sawada (2001) found particular mRNAs to be highly enriched in the vegetal hemisphere

OneoftheseRNA messages encodes a zinc­ finger transcription factor called Macho-I

concentrated the vegetal hemisphere of the unfertilized egg and remains present during early fer­ tilization. It appears to migrate with the yellow crescent cytoplasm into the posterior vegetal region of the egg dur­ ing e second half of the first cell cycle. By the 8-cell stage, ma -l A is found only in the B4.1 blastomeres. At the 16- and 32-cell stages, it is seen those blastomeres that give rise to the muscle cells

When antisense oligonucleotides to deplete macho-l mRNA were injected into unfertilized eggs, the resulting ae Jacked the muscles usually fo ed by the descen­ dants of the B4.1 blastomere. (They did have the second­ ary muscles that are generated through the interac ons of A4.1 and b4.2 blastomeres.) The tails of these ma -l­ depleted larvae were severely shortened, but the other regions of the tadpoles appeared structurally and biomem-
(D)
(E)
was fo d to be concentrated the vegetal hemisphere of the unfertilized egg and remains present during early fer­ tilization. It appears to migrate with the yellow crescent cytoplasm into the posterior vegetal region of the egg dur­ ing e second half of the first cell cycle. By the 8-cell stage, ma -l A is found only in the B4.1 blastomeres. At the 16- and 32-cell stages, it is seen those blastomeres that give rise to the muscle cells’

B4.1 blastomeres isolated from 1 1cho-I-depleted embryos failed to produce muscle tissue.

cells that would not normally fonn muscle, and found that these ectoderm or endoderm precursors did generate muscle cells when given macho-I mRNA.

transcription factor that is required for the activation of several mesodermal genes, including muscle acHl1, myosin. tbx6, and snail

only the Tbx6 protein produced muscle differentiation (as Macho-l did) when expressed in cells ectopically.

Macho-I proteiny is both necessary and sufficient to promote muscle different ascidian cells

snail protein is important in preventing Brachyury expression in presumptive muscle cells, and is therefore needed to prevent the mus­ cle precursors from becoming notochord

Macho-l activates a transcription factor cas­ cade that promotes muscle differentiation while at the same time inhibiting notochord specification.

The posterior cells that will become mesenchyme respond differently to the FGF signal due to the presence of Macho-1 in the posterior vegetal cytoplasm (Figure 5.39; Kobayashi et aL 2003). Macho-1 prevents notochord induc­ tion in the mesenchymal cell precursors by ac va ting the snnil gene (which will in turn suppress the activation of 8rachyllry

Macho-l is not only a muscle-activating determinant, it is also a factor that distinguishes cell sponse to the FGF Signal. These FGF-responding cells do not become muscle, because FGF also activates cascades that block muscle formation