1. Early Development
Amphibians

2. Cleavage in a frog Describe the progression of events.
differences in size of blastomeres
morula and blastula stages; presence of blastocoel
cell held together by EP-cadherin

3. Gastrulation in Xenopus
What have fate maps told us about tissue origins?

4. Gastrulation in Xenopus
What are VegT and Vg1 and what influcence do they have?
VegT – transscription factor; Vg1 – paracrine factor;mRNA’s located in cortical cytoplasm of unfertilized egg
needed for prospective fate development

5. Gastrulation in Xenopus
What type of cell movement initiates gastrulation?
invagination of cell in grey crescent region on future dorsal side; shape chages, formation of bottle cells

6. Gastrulation in Xenopus
What happens next?
involution of marginal zone cells; epiboly of animal pole cells
always new cells at dorsal lip; involution of marginal zone cells
first cells are cells that become pharyngeal cells, then precursor of head mesoderm, then chordomesoderm
get displacement of blastocoel, formation of archenteron; involution of lateral and ventral lips; yolk plug

7. Gastrulation in Xenopus
What happens at the mid-blastula transition in preparation for gastrulation?
How is dorsal-ventral axis established?
What happens if cortical rotation is blocked?
demethylation of certain promoters; allows transcription factors like VegT to initiate new transcription
activation of zygote genome
dorsal side forms 180 degrees from sperm entry site; blastopore
no dorsal development
rotation of egg can change site of gastrulation

8. Gastrulation in Xenopus
How did Gimlich and Gerhart demonstrate that certain cells were responsible for inducing dorsal lip formation?

9. Gastrulation in Xenopus
What appears to be the major factor directing movement of cells into embryo?
vegetal rotation – superficial layer of marginal cells is pulled inward because it is attached to actively migrating deep cells

10. Gastrulation in Xenopus
What other cell movements occur?
intercalation of IMZ leading to convergent extension

11. Gastrulation in Xenopus
What two factors appear to drive convergent extension?
adhesion proteins paraxial and axial protocadherin are expressed
changes in calcium concentrations

12. Gastrulation in Xenopus
At what point in gatrulation do notocordal cells become distinct?
toward the end – perhaps because of different adhesion molecules

13. Gastrulation in Xenopus
What mechanism appears to be responsible for epiboly of the ectoderm?
increase in cell number and intercalation of several layers of cells

14. Axis Formation
How did Hans Spemann first demonstrate the importance of the grey crescent?
newt –Triturus taeniatus

15. Axis Formation
What did Spemann do to demonstrate changes in cell potency during gastrulation?

16. Axis Formation
What classic experiment was done by Spemann and Mangold that led to the concept of the organizer?
Triturus taeniatus and Triturus cristatus
called primary embryonic induction

17. Axis Formation
How did work of Nieuwkoop and Nakamura contribute to an understanding of how the “organizer” worked?
combined animal and vegetal caps to generate mesodermal tissue

18. Axis Formation What is the Nieuwkoop center? How is it formed?
dorsalmost vegetal cells capable of inducing the organizer
created by cytoplasmic rotation at fertilization

19. Axis Formation
What is involved in the mechanism of induction of mesoderm by endoderm?
What appears to be the role of β-catenin?
What is the connection between β-catenin and glycogen synthase kinase – 3 (GSK-3)?
endodermal cells secrete activin like (derriere) factor that causes expression of Brachyury gene in mesodermal cells
Brachyury protein acts as a transcription factor to induce eFGF that maintains Xbra expression and activates genes that produce mesodermal specific proteins
factor forming Nieuwkoop center; key player in formation of dorsal structures
GSK-3 negatively regulates β-catenin

20. Axis Formation
How does β-catenin become concentrated in dorsal cells of embryo?
initially made throughout embryo; degraded by GSK-3 in ventral cells
disheveled, an inhibitor of GSK-3 is translocated to doral side during cortical rotation

21. Axis Formation
How does β-catenin contribute to formation of the organizer?
TGF-β factor may be Vg1 or nodal proteins

22. Axis Formation
What model was developed by Agius to explain mesodermal induction?

23. The Organizer What are the four major functions of this tissue?
differentiate into dorsal mesoderm
induce formation of paraxial mesoderm
induce formation of neural tube
initiate gastrulation movements

24. The Organizer What is the role of the goosecoid gene?
Abilitiy of goosecoid to induce new axis
a)uninjected embryo – one dorsal lip
b) injected with goosecoid into ventral vegetal blastomeres – two dorsal lips
c) top embryos show dorsal axis
d) twinned embryo from injection
gene codes DNA binding protein that activates migration properties of cells, determines fate of dorsal mesodermal cells, recruits neighboring cells into dorsal axis, activates a gene needed for brain formation
goosecoid works in nucleus of cells

25. The Organizer
How does the organizer actually induce formation of neural tissue?
induction actually lead to formation of epidermal tissue from ectoderm – via BMP
organizer secretes molecules that block induction – get default neural tissue

26. The Organizer
What are the proteins secreted by the organizer?

27. The Organizer What can the secreted protein noggin do?
What other proteins exhibit similar effects?
rescue UV irradiated embryos
induces dorsal ectoderm to form neural tissue and dorsalizes mesodermal cells
binds to BMP-2 and BMP-4 and prevents their binding to receptors
chordin and nodal-related 3, follistatin

28. The Organizer What are some of the important effects of BMP-4?
activates expression of genes encoding transcription factors that induce expression of epidermal and ventral mesodermal differentiation – suppress genes that produce neural phenotype
low dose causes muscle to form; intermediate dose causes kidney cells; high dose causes blood cells to form

29. The Organizer
What are the functions of the diffusable proteins cerberus, dickkopf and Frzb?
How do these proteins work?
secreted by pharyngeal endoderm and prechordal mesoderm and induce most anterior head structures
block wnt pathway and BMP-4

30. The Organizer

31. The Organizer Why is neurogenin so important?
produces neural phenotype

32. Induction
How did Otto Mangold demonstrate the regional specificity of neural induction?
portion of archenteron roof transplanted determined neural structures that formed

33. Induction
Toivonen and Saxen postulated two gradients in amphibian embryos
dorsal gradient – “neuralizing” activity
caudal gradient – “posteriorizing” activity
What is the primary protein involved in posteriorizing the neural tube?
Xwnt8 protein – can add to developing embryos in anterior region and alter course of development; can suppress wnt signaling and get more neural cells
BMP specifies dorsal-ventral axis; wnt specifies anterior-posterior axis
other factors like FGF and retinoic acid may be involved in a-p axis

34. Induction What positive signal promotes anterior head development?
insulin-like growth factors

35. Left-Right Axis
What appears to be major event in left-right axis formation in Xenopus?
expression of Xnr1 in lateral plate mesoderm on left side
may be initiated at fertilization via Vg1 protein on left side