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What are the stages of embryonic development?

- Haploid --> diploid; generates diversity (genome mixing); completion of meiosis

Cleavage divisions:
- rapid increase in cell number (mitosis but very short G phases)
- often zygote starts gene expression, often cells become specified for different fates

- extensive cell movements and re-organisation of cells into germ layers, inductive interactions, body axes become evident
- many tissues become specified and differentiate along different lineages
- to be specified means a cell has received enough signals to follow a certain fate, but it doesn't mean it is committed
- if you take that cell and put it in vitro it will follow along that fate
- doesn't mean this isn't reversible
- if you take that cell in vitro and give it certain signals you can drive it down another fate
- determined = cell has actually started down its fate, almost irreversible, put it in a foreign environment will continue in this determined fate regardless of other signals

- further inductive interactions between different tissues
- tissues differentiate into organs


What are the four vertebrates species commonly used in developmental biology research? What are differences and similarities?

- xenopus - big egg
- zebrafish - medium size egg
- chick - huge egg
- mouse - tiny egg

- vertebrates have very different sized eggs, patterns of early development and gastrulation
- yet can appear very similar during organogenesis (phylotypic stage)
- mature animal: very different shapes but body plans are very similar

- develop into blastoderm (chick) /blastocyst (mouse) /blastula (xenopus, zebrafish)
- this is the time after all the cleavage events have happened and about the time that gastrulation is about to commence, formation of the germ layers

- dorsoventral, left-right
- body plan laid down after gastrulation
- hard to tell mouse and human embryo apart
- mouse embryo has smaller head and smaller tail


How does cleavage occur in xenopus?

- after fertilisation (~90 minutes) first cleavage (20' intervals) --> blastula (~1k cells around blastocoel)
- rapid rate of proliferation
- no growth between divisions (maternal mRNA/proteins)
- vegetal cells larger than animal cells - (yolk affects cleavage)
- yolk tends to be an inhibitor of cleavage divisions: animal cap has less yolk, vegetal has lots
- growth phases in between are almost non-existant hence fast cleavage stages
- no growth of the embryo --> cells just decrease in size
- still undergoing meiosis/cytokinesis: ratio of cytoplasm to nucleus/DNA getting smaller
- decrease in ratio is one of the key things that drives zygotic gene expression
- doesn't have enough mRNA so zygotic DNA starts to be expressed
- mid-blastula transition (MBT) - zygotic gene expression commences in late blastula (DNA:cytoplasm ratio), some genes at 256 cell stage


How does cleavage occur in the zebrafish?

- cleavage divisions do not extend into yolk; blastomeres sit on the yolk
- very different pattern of cleavage divisions to xenopus
- blastoderm (~1000 cells lying over yolk)
- zygotic expression @ ~512 cell stage

- very rich in yolk - more yolk than xenopus and as a result cleavage divisons are slower and tend to be less complete
- accumulation of cells on the animal cap part of the embryo whereas the yolk region of the embryo rarely ever undergoes cleavage


What is the importance of yolk?

- yolk affects cleavage divisions
- different levels of yolk will affect how the cleavage of different species occurs


How does cleavage occur in the chick?

- cleavage divisions incomplete (yolk)
- blastoderm (~60k cells lying over yolk and has a sub-germinal space)
- zygotic gene expression detectable in early blastoderm
- yolk = oocyte
- cleavage furrow where cytoplasm is
- doesn't fully partition til about 30-40 or even 100 cells when the cleavage divisions start to become much more complete
- cleavage divisions start to become much more complete as the cells start to separate more frequently (separated by sub-germinal space)
- two lineages detectable in the early blastoderm - epiblast and hypoblast --> two very different lineages in animal
- preceeds gastrulation
- get replaced during gastrulation
- zygotic gene expression already switched on - epiblast slightly different to hypoblast


How does cleavage occur in the mouse?

- ~24 hr after fertilisation, complete cleavage (no yolk) divisions every 12 hours
- morula - compacts, lumen forms, leading to blastocyst (~16-32 cell stage)
- very early on you get zygotic gene expression: cadherin molecules, these facilitate compaction
- blastocyst - two different cell types (trophectoderm and inner cell mass)
- cell fates in blastocyst specified:
-- ICM - embryo proper
-- trophectoderm - extraembryonic membranes/placenta
- fate of ICM cells not specified (mary's lecture on pluripotency and KOs)


What is a blastomere?

- individual cell in an embryo still undergoing cleavage


What is cell fate mapping?

- question of when the fates of cells become specified
- first embryo that was well identified in terms of fate mapping was xenopus
- animal cap cells
- larger vegetal cells
- injected each embryo, charcoal markers of GFP
- painstaking

- can we determine if cells have a certain fate?
- xenopus, late blastula cell fates mapped
- ectoderm (animal cap region; endoderm vegetal regions)
- future mesoderm originates in band of cells between animal and vegetal poles
- pretty much all the cells have been specified in late blastula
- ventral to dorsal in future mesoderm: blood, kidney/ somites, heart/ notochord
- ventral to dorsal animal cap: epidermis / nervous system
- mesoderm already segmented into very specific structures


What is the dorsal view of fate mapping?

- despite differences in cleavage and development
-- cell fates (ectoderm, mesoderm, endoderm) specified by late blastula/blastoderm stages (prior to gastrulation) in various species
- dorsal-ventral axis is evident (dorsal structures: neural ectoderm, notochord)
- anterior-posterior axis is evident
- how do body axes arise?


What are the body axes?

- how does the embryo develop a front-back axis?

- how does the embryo develop a head-tail axis?

L-R asymmetry
- how does an embryo develop a left-right axis?


How does the animal vegetal axis develop in the embryo?

- before fertilisation, the animal pole clearly different from the vegtal pole (animal pole pigmented)
- maternal determinants (mRNAs, proteins) differentially localised along the animal/vegetal axis
- Vg1 (TGF-beta family of growth factors), Veg-T (transcription factor), XWnt11 mRNA localised to the vegetal cortex of the oocyte

- Veg-T localised in vegetal pole, very little in the animal pole

- but what relation does this have to body axes?


How does sperm entry affect D-V axis in xenopus?

- dorsal side of the embryo develops opposite the site of sperm entry
- formation of grey crescent (mixing of vegetal and animal cytoplasm – pigment)
- fertilisation induces cortical rotation of cytoplasm


What are the Wnt/Beta-catenin signals in relation to the D-V axis?

- cortical rotation results in re-location of componets of the Wnt pathway (Dvl, Wnt11 to dorsal side of the emrbyo)
- activation of Wnt signalling results in accumulation of Beta-catenin in nuclei of dorsal blastomeres

- so fertilisation and cortical rotation shifts maternal determinants, and they drive activation of the wnt/beta-catening signalling pathway

- cortical rotation of maternal determinants (Dvl, Wnt11, Beta-catenin) induces expression of zygotic genes via activation of Wnt signalling in dorsal blastomeres
- common to many species (frog, fish, birds, mouse)
- zenopus - presence of beta-catenin in vegetal cells indicates 'Nieuwkoop Centre'


What are the wingless signals?

- frizzled receptor
- recruit dishevelled when Wnt binds
- scaffold in the cytoplasm: axin, APC, CK1 (gilgamesh), GSK-3 (zeste-white)
- normally degrade beta-catenin bound ot APC, but when dishevelled is recruited by wnt binding frizzled, beta-catenin is freed from scaffold and moves into nucleus to act as a transcription factor


How does gravity relate to development of D-V axis in chick?

- rotation (every 6 min) of chick egg during passage in the oviduct results in asymmetric distribution of maternal determinants (driven by gravity) (rotations caused by smooth muscle acting in the oviduct)
- posterior marginal zone cells form at highest point of tilted blastoderm; express Wnt8c and Vg1; defines D-V axis
- PMZ leads to formation of primitive streak - gastrulation and anterior-posterior axis


What do maternal determinants do in the xenopus embryo?

- asymmetric distribution of maternal determinants leads to activation of Wnt/Beta-catenin pathway and induction of dorsal genes (D-V axis)
- but this process is inter-linked with mesoderm induction and specification of anterior-posterior (A-P) axis


Where does mesoderm come from?

cell culture experiments:
- marginal zone cells form mesodermal tissues
- dorsal mesoderm and ventral mesoderm are already specified
- dorsal cells --> dorsal mesoderm tissues
- ventral cells --> ventral mesoderm tissues

- vegetal cells induce animal cap cells to become mesoderm

- if you take animal cap cells and combine them with the vegetal cells and put them in vitro
- animal cap cells are induced by vegetal cells to become various mesodermal structrues


what happens if you take cells from the late blastula of xenopus?

- animal cap cells --> ectoderm
- ventral marginal zone: mesoderm, mesenchyme, epidermis, blood
- dorsal zone: mesoderm, notochord, muscle, neural tube
- vegetal cells: undifferentiated vegetal tissue - not yet specified


Do dorsal and ventral vegetal cells have different properties?

dorsal vegetal cells
- dorsal mesoderm
- notochord
- muscle

Ventral vegetal cells
- ventral mesoderm
- blood
- mesenchyme

- inducing properties of ventral and dorsal vegetal cells are different?
- why?


What are mesoderm inducers?

maternal determinants in vegetal cells
- Vg1 (TGF-beta family of growth factors)
- Veg-T (transcription factor)

Earliest zygotic genes expresses in vegetal cells
- xnr5, xnr6, derriere (nodal-related proteins, TGF-beta family)

- deplete Veg-T --> failure of XNr expression and mesoderm formation
- rescue mesoderm formation by injected mRNA for Xnr proteins