lecture 30

Question 1

What are the stages of embryonic development?

Answer 1

Fertilisation:
- 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

Gastrulation

• 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

Organogenesis

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

Question 2

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

Answer 2

• 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

Question 3

How does cleavage occur in xenopus?

Answer 3

• 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

Question 4

How does cleavage occur in the zebrafish?

Answer 4

• 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

Question 5

What is the importance of yolk?

Answer 5

• yolk affects cleavage divisions

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

Question 6

How does cleavage occur in the chick?

Answer 6

• 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

Question 7

How does cleavage occur in the mouse?

Answer 7

• ~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)

Question 8

What is a blastomere?

Answer 8

• individual cell in an embryo still undergoing cleavage

Question 9

What is cell fate mapping?

Answer 9

• 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

Question 10

What is the dorsal view of fate mapping?

Answer 10

• 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?

Question 11

What are the body axes?

Answer 11

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

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

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

Question 12

How does the animal vegetal axis develop in the embryo?

Answer 12

• 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?

Question 13

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

Answer 13

• 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

Question 14

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

Answer 14

• 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’

Question 15

What are the wingless signals?

Answer 15

drosophila

• 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

Question 16

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

Answer 16

• 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

Question 17

What do maternal determinants do in the xenopus embryo?

Answer 17

• 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

Question 18

Where does mesoderm come from?

Answer 18

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

Question 19

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

Answer 19

specification

• 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

Question 20

Do dorsal and ventral vegetal cells have different properties?

Answer 20

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?

Question 21

What are mesoderm inducers?

Answer 21

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)

Experiments

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