Patterning Amphibian Embryo Flashcards
How is first axis set up by sperm entry into egg
Rotation of cortical (surface) cytoplasm near vegetal pole away from sperm entry point with respect to inner parts
Sperm entry point will become ventral side
First region of cell sheet to tuck in
Occurs after zygotic trabscription occurs
The point of the blastula that will become the dorsal side of embryo begins tucking in so start gastrulation
Xenopus blastula fate map
On diagrams
Animal vegetal axis
Ectoderm near animal
Middle band becomes mesoderm
Notochord by future dorsal
Ventral side of hand becomes blood and kidney
In between two becomes somites
Nervous system from f dorsal roo
Endoderm from vegetal side
Corresponding in larva on diagrams too
Patterning mesoderm blastula experiment
Take blastula
Remove animal cap and culture it
Will only make epidermis no nervous system
Culture bottom part - will only make endoderm cells that surround the gut
Remove middle band (future mesoderm) and just graft animal and vegetal poles together
If done early enough patterning in the embryo can be fully restored just a bit runty due to missing mass
The mesoderm cell types did not form in the animal and vegetal only cultures
But arise from animal cap cells when the animal and vegetal sides are together
Signals from vegetal Must be from maternal gene products as zygotic transcription starts later than early blastula
The dorsoventral orientation of the mesoderm conforms to the orientation of the vegetal lump
Suggests they signals from the vegetal lump are different for dorsal and ventral sides
Dorsal ventral patterning experiment
Took early gastrula when gastrulation just beginning
Take dorsal mesoderm and graft it to opposite side of host embryo
This grafted dorsal tissue causes the dorsal side to be mirrored
Dorsal mesoderm tissue goes on to form notochord
Which then signal to surrounding tissues to become other dorsal tissues
Induced patterning
Only the dorsal tissue has this long range patterning effect on neighbours
Can argue that there are signals produced in dorsal tissue that affect both mesoderm patterning snd patterning of the ectoderm above it
Eg new nerve cord above the ectopic notochord
Zygotic transcription has begun in early gastrula so can assume these are zygotic patterning factors
Identifying the maternal vegetal determinants
Look for RNAs present only in the vegetal pole
Veg1 RNA localised strongly in vegetal side of blastula
Maternal gene product added in oogenesis (preferrilisation)
Localised vegetally
Is a member of the TGF signalling pathway
Unsure how the oocyte is polarised and how the Vg1 ends up there (unlike drosophila oocyte polarisation)
Remains in vegetal region for long while
Identifying maternal Vg1 function
RNA knockdown (can’t feasibly directly manipulate gene)
Inject antisense rna to Vg1 RNA and prevent its function
Vg1 KO caused v reduced mesoderm
No notochord
V reduced nervous system
Vg1 part of vegetal signalling from earlier
But doesn’t work alone here
Another maternal product - VegT TF localised vegetally also gives similar KO phenotype
Establishing the dorsal side
Through stabilising beta catenin
Sperm enter egg
Certain amount of cytoskeleton comes with it
Introduction of the small amount of MTs induces rotation
Almost as if the MTs elongate to the shear zone (zone between inner and outer cytoplasm)
Causes rotation of the outer cytoplasm
The MTs elongate out to periphery
When teach shear zone the MT tips are deflected and have their elongating plus ends around away from sperm entry site
They line up
These MTs transport maternally produced gene product Dishevelled - associated with lines in and moves toward plus end
Also moves Wnt11 RNA
Dishevelled stabilises beta catenin which takes part in a TF complex (this is still at single cell stage)
Maternal beta catenin is throughout whole egg after laying
But it is only stabilised by dishevelled that has been moved to the future dorsal side so is only stably present dorsally
How does the dorsal beta catenin affect the embryo
Via zygotic transcribed vegetal signal
Consequence if one of the earliest zygotic gene transcription events
Only transcribed in the yolky vegetal cells
Nodal transcription activated by Vg1 and VegT
Stable Beta catenin upregulates NODAL transcription in early-mid blastula
So causes a gradient in vegetal side of embryo highest in dorsal end
Consequence of stronger nodal signalling in Dorsal vegetal pole
Stronger at dorsal
Weaker at ventral
The animal cap contacts the vegetal pole
Signalling from vegetal pole turns on CHORDIN bad X BRA transcription in these cells
These are mesoderm only markers so vegetal nodal has induced change in animal cap
Inhibiting nodal production inhibits chordin and x bra
Mesoderm induction and patterning
Strong nodal signalling + beta catenin presence
Turns on SIAMOIS TF
Does 2 things relevant to BMP signalling
-blocks BMP production in SIAMOIS synthesising cells
-also activated secreted BMP antagonists
>chordin
>noggin
This causes a gradient of BMP across the embryo
Strong BMP at ventral
Weak BMP at dorsal - due to the BMP sink activity
Opposite direction to the NODAL and beta catenin gradient
BMP gradient function
Adding more bmp
Or blocking bmp
Makes uninteroretable mess
But can take a piece of the embryo and look to see what cell types arise from it in culture
Ventral tissue doesn’t form notochord
But as BMP is blocked more and more
Notochord tissue starts to form
Ventral side usually forms blood so as BMP is inhibited blood production drops
As BMP increases
Notochord formation drops
Blood formation up
Chordin/noggin gradient
Secreted proteins
Bind BMP4 and block function
Produced by dorsal cells due to max level of nodal signalling and beta catenin
Are Secreted and form gradient across DV axis from dorsal
Antagonises BMP
So causes BMP gradient running in the opposite direction V-D
Anatomy of amphibian late embryo
Mesoderm
-notochord - most dorsal
-somites
-lateral plate
-blood - most ventral
AP axis patterning signals basic
WNT signal produced in posterior
WNT antagonists produced at anterior
WNT gives posterior identity
