1. Amphibians & Fish Early Development and Axis Formation
Chapter 7 – Part 1

2. Amphibian and Fish Backbone of vertebrate embryology  Frogs (Xenopus)
Large cells and rapid development
But, long period before fertile
Zebra fish (Danio rerio)
Model organism for study of vertebrate development
Breed each year
Easily maintained
Transparent embryos
At 24 hours after fertilization – embryo has formed most of its organ primordia

3. Fertilization, Cortical Rotation, and Cleavage
Fertilization and cortical rotation
Can occur anywhere in animal hemisphere of amphibian
Sperm entry point determines orientation of the dorsal ventral axis
Marks ventral side
180° opposite sperm entry point marks the dorsal side
Centrioles organize microtubules in egg
Arrange in parallel array in vegetal cytoplasm
Cortical cytoplasm Rotates 30° relative to internal cytoplasm
Array disappears when rotation stops

4. Fertilization, Cortical Rotation, and Cleavage
7.1 Formation of the parallel arrays of microtubules in the vegetal hemisphere along the future dorsal-ventral axis
40% first cell cycle complete – microtubules begin forming
50% first cell cycle complete – more microtubules, but lack polarity
70% first cell cycle complete – parallel array of microtubules

5. Fertilization, Cortical Rotation, and Cleavage
Opposite sperm entry site
Gray crescent
Where gastrulation will begin
7.1 Reorganization of cytoplasm in the newly fertilized frog egg

6. Fertilization, Cortical Rotation, and Cleavage
7.1 Reorganization of cytoplasm in the newly fertilized frog egg

7. Unequal Radial Holoblastic Cleavage
Radially symmetrical and holoblastic
Contains more yolk
Concentrated in vegetal pole
1st cleavage bisects gray crescent
2nd cleavage starts before 1st finishes
Uneven division due to presence of yolk in vegetal hemisphere
Even with unequal size
Blastomeres continue to divide at same rate

8. Unequal Radial Holoblastic Cleavage
7.2 Cleavage of a frog egg

9. Unequal Radial Holoblastic Cleavage
7.3 Scanning electron micrographs of frog egg cleavage
First cleavage
Second cleavage (4cells)
Fourth cleavage (16 cells)

10. Unequal Radial Holoblastic Cleavage
Unequal radial holoblastic cleavage (cont’)
Small micromeres at animal pole
Large macromeres at vegetal pole
Regulated by MPF (mitosis promoting factors)
At cell stage
called MORULA (mulberry)

11. Unequal Radial Holoblastic Cleavage
At 128 cell stage
Blastocoel formed
Embryo considered a Blastula
2 functions:
Permit cell migration during gastrulation
Prevent cells beneath it from interacting prematurely with cell above
Roof of blastocoel in animal hemisphere
Animal cap
If placed near bottom of vegetal cells – cap cells become mesoderm
Cleavage to Blastula

12. Unequal Radial Holoblastic Cleavage
Mesoderm formed next to vegetal endoderm precursors
Animal cap destined to become nerves and skin
Cell adhesion molecules keep cells together
EP cadherin
7.4 Depletion of EP-cadherin mRNA in the Xenopus oocyte results in the loss of adhesion between blastomeres and the obliteration of the blastocoel

13. Mid Blastula Transition: Preparing for Gastrulation
Very few genes transcribed during early cleavage
Nuclear genes are not activated until the 12th cell cycle
Acquires capacity to become motile
Mid blastula transition (MBT)
Egg factors initiate this (absorbed by newly made chromatin)
Different genes activated in different cells
Cell cycle acquires gap phases
Blastomeres acquire capacity to become motile

14. Amphibian Gastrulation
Multiple ways of gastrulation in different amphibians
Xenopus fate map
Goal is to bring inside embryo areas destined to form endodermal organs
Blastula has different fates depending on whether a cell is located deep or superficially in the layers
Mesoderm precursors exist mainly in deep layers
Endoderm (bottom) and Ectoderm (top)

15. Vegetal Rotation and the Invagination of the Bottle Cells
Gastrulation movements in the frog
To position the mesoderm between the outer ectoderm and the inner endoderm
Vegetal rotation and the invagination of the bottle cells
Gastrulation starts on the future dorsal side of embryo
Below equator
Region of gray crescent
Region opposite the sperm entry point

16. Amphibian Gastrulation
7.6 Cell movements during frog gastrulation

17. Vegetal Rotation and the Invagination of the Bottle Cells
At this point, cells invaginate to form a slitlike blastopore called bottle cells
Change shape
Maintain contact with outside cells
Bottle cell’s line the archenteron
Similar to sea urchins except gastrulation in frogs begins in marginal zone not ventral region
Endoderm not as large/yolky here as other vegetal blastomeres

18. Vegetal Rotation and the Invagination of the Bottle Cells
Experiments (Hardin & Keller) proved:
Dorsal lip cell removed placed on inner endoderm
Formed bottle cells and sank below the surface
Once involution starts
Gastrulation will proceed without bottle cells
Coordinated involution of subsurface cells more important

19. Vegetal Rotation and the Invagination of the Bottle Cells
Initiated by internalization of endoderm and mesoderm
2 hours before bottle cells are formed
Rotation puts prospective pharyngeal endoderm adjacent to the blastocoel and above involuting mesoderm
Marginal cells form endodermal lining of archenteron
If the involuting marginal zone (IMZ) cells were removed
Archenteron formation would stop

20. Vegetal Rotation and the Invagination of the Bottle Cells
7.8 Early movements of Xenopus gastrulation

21. Involution at the Blastopore Lip
Involution of blastopore lip
Involution of marginal zone cells
Animal cells undergo Epiboly and converge at blastopore
Dorsal lip of blastopore
Migrating Margin cells turn inward and travel along inside animal hemisphere
Form the lip of the blastopore
Constantly changing

22. Amphibian Gastrulation
7.6 Cell movements during frog gastrulation

23. Amphibian Gastrulation
7.6 Cell movements during frog gastrulation

24. Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
7.7 Surface view of an early dorsal blastopore lip of Xenopus

25. Involution at the Blastopore Lip
Dorsal lip of blastopore
Margin cells turn inward and travel inside animal hemisphere
Form the lip
Will become cells that involute to form prechordal plate
Precursor of the head mesoderm
Followed by next cells to involute in at lip to become chordamesoderm
Will form the notochord
Important to inducing and patterning nervous system

26. Amphibian Gastrulation
7.8 Early movements of Xenopus gastrulation

27. Involution at the Blastopore Lip
Slowly blastocoel is displaced to opposite side of the dorsal lip
Lateral lips and ventral lips form on “crescent” to make additional mesoderm and endodermal cells
Finally a ventral lip covers over additional mesodermal and endodermal precursor cells
Forming a “ring” blastopore
Remaining endoderm patch is called the yolk plug
Eventually internalizes

28. Amphibian Gastrulation
7.9 Epiboly of the ectoderm

29. Convergent Extension of the Dorsal Mesoderm
Involution begins dorsally
Pharyngeal endoderm and head mesoderm
Next tissue to enter forms the notochord and somite
Lip of the blastopore expands to have dorsolateraly, lateral, and ventral sides
Prospective heart, kidney, and ventral mesoderm that enters the embryo

30. Convergent Extension of the Dorsal Mesoderm
Blastopore lip IMZ cells (involuting marginal zone)
Several layers deep IMZ cells intercalate radially
Continues to extend vegetally
As these cells reach blastopore lip – involute inwardly initiation 2nd intercalation
Causes convergent extension along mediolateral axis
Simultaneous elongation with involution
Mesoderm continues to migrate to animal pole
Forms an endodermal roof of the archenteron

31. Convergent Extension of the Dorsal Mesoderm
7.10 Xenopus gastrulation continues

32. Convergent Extension of the Dorsal Mesoderm
Central Dorsal mesoderm
Become notochord and somites
Remainder of body mesoderm
Forms heart, kidneys, bones, …..
Entered through ventral and lateral blastopore lips
These create the mesodermal mantle
Endoderm formed from involuting marginal zone (IMZ) that forms lining of archenteron roof
Endoderm from vegetal cells that become archenteron floor
Where endoderm meets ectoderm becomes anus

33. Epiboly of the prospective ectoderm
The animal cal and noninvoluting marginal zone (IMZ) cells expand by epiboly
Covers the entire embryo
Form the surface ectoderm
By increasing the cell number (through division) coupled with concurrent integration of several deep layers into one
Involves assembly of fibronectin into fibrils

34. Epiboly of the prospective ectoderm
7.13 Epiboly of the ectoderm is accomplished by cell division and intercalation

35. Progressive Determination of the Amphibian Axes
Remember
This process whereby the central nervous system forms through interactions with the underlying mesoderm
Called Primary Embryonic Induction
A principle way vertebrate body becomes organized
Discoverers called the dorsal blastopore lip
“The Organizer”

36. Hans Spemann and Hilde Mangold: Primary Embryonic Induction
Began his most famous work in 1903 on demonstrating nuclear equivalence
Later he won the Nobel Prize in 1935
Later his work (1924) centered on the dorsal lip cells, referred to as the Spemann’s Organizer
Hilde Mangold
PhD Student of Spemann
Began assisting him in his work in 1924 and contributed to understanding cell fate in early gastrulation
These cells originated as self-differentiating tissue of the dorsal lip
Derived from the gray crescent cytoplasm

37. Hans Spemann and Hilde Mangold: Primary Embryonic Induction
Spemann Organizer
Role of the dorsal lip cells destined to be:
Dorsal mesoderm
Notochord
Some anterior pharyngeal
Induce host’s ventral tissue to change fate and form neural tube and dorsal mesoderm (such as somites)
Organize host and donor tissues into 2nd embryo with AP and DV axes using donor tissue

38. Hans Spemann and Hilde Mangold: Primary Embryonic Induction
7.17 Organization of a secondary axis by dorsal blastopore lip tissue

39. Hans Spemann and Hilde Mangold: Primary Embryonic Induction
Spemann Organizer (cont)
Responsible for neural tube formation
Chordamesoderm & ectoderm cannot organize the entire embryo
Responsible for transforming flanking mesoderm into AP axes
These events initiate a sequential series of sequential inductive events
This is Key Inductive Event
Dorsal lip cells inducing the A-P axis and neural tube
Called Primary Embryonic Induction

40. Molecular Mechanisms of Amphibians Axis Formation
This actually created more questions than answers by these experiments
How does the organizer form?
~ dozen cells of the initial organizer position themselves opposite the point of sperm entry
Now discovered that they are in the right position for 2 signals converging
First signal – tells the cells that they are dorsal
Second signal – tells them they are mesoderm

41. The Dorsal Signal: -Catenin
Two signals & others:
Dorsal signal: -caterin
Isolating cap, marginal, vegetal cells
Organizer cells are special because they reside above a special group of vegetal cells
Recombined animal cap with vegetal cells
Vegetal cells induced mesoderm formation from animal cap cells (marginal/equatorial cells)
Ventral vegetal – blood, mesenchyme
Intermediate vegetal – muscle, kidney
Dorsal vegetal – somites, notochord
These dorsal inducing vegetal cells called the Nieuwkoop center

42. The Dorsal Signal: -Catenin
7.18 Summary of experiments by Nieuwkoop and by Nakamura and Takasaki, showing mesodermal induction by vegetal endoderm

43. The Dorsal Signal: -Catenin
β-caterin
Activin-like TGF-β &FGFs
7.18 Summary of experiments by Nieuwkoop and by Nakamura and Takasaki, showing mesodermal induction by vegetal endoderm

44. The Dorsal Signal: -Catenin
Proof of dorsal vegetal cells is inducer of organization
Nieuwkoop center
Similar proof of dorsal-most vegetal cells induce animal cell to form dorsal mesoderm
-caterin
Protein acting as cell anchor for cadherins
Found to give this property to dorsal vegetal cells
[In sea urchin responsible for specifying micromeres]

45. The Dorsal Signal: -Catenin
If -catenin is originally found throughout the embryo, how does it become localized specifically to the side opposite the sperm entry?
Answer  translocation of Wnt11 and the Disheveled (Dsh) protein from the vegetal pole to the dorsal side of the egg at fertilization
-catenin is targeted for destruction by glycogen synthase kinase 3 (GSK3)
GSK3 destroys b-catenin and blocks axis formation
GSK3 is inactivated by GSK3-binding protein (GBP)
Occurs during the 1st cell cycle when microtubules are formed

46. The Dorsal Signal: -Catenin
7.21 Model of the mechanism by which the Disheveled protein stabilizes -catenin in the dorsal portion of the amphibian egg

47. The Dorsal Signal: -Catenin
GSK3-binding protein (GBP)
Travels along the microtubules by binding to kinesin
Moves to a point opposite sperm entry
Disheveled grabs the GPB and is translocated on microtubules
Cortical rotation is probably in orienting and straightening the microtubular array
GBP and Dsh are released at the opposite point of sperm entry
Future Dorsal side of embryo
They inactivate GSK3  allowing -catenin accumulation
Ventral side GSK3 is degraded

48. The Dorsal Signal: -Catenin
7.20 The role of Wnt pathway proteins in dorsal-ventral axis specification, -catenin (orange)

49. Function of the Organizer
Nieuwkoop center cells remain endodermal, cells of the organizer become dorsal mesoderm and migrate underneath dorsal ectoderm
Dorsal mesoderm induces central nervous system formation
Properties of organizer – 4 major functions
Ability to self differentiate dorsal mesoderm
Ability to dorsalize surrounding mesoderm into paraxial mesoderm (somites)
Ability to dorsalize ectoderm forming neural tube
Ability to initiate the movement of gastrulation

50. Function of the Organizer
Organizer cells ultimately contribute to 4 cell types:
Pharyngeal endoderm
Head mesoderm (prechordal plate)
Dorsal mesoderm (primarily notochord)
Dorsal blastopore lip
1 & 2 – induce forebrain and midbrain
3 – induces hindbrain and trunk
4 – remains to end of gastrulation then becomes chordaneural hinge that induces the tip of the tail

51. Induction of Neural Ectoderm and Dorsal Mesoderm: BMP Inhibitors
Problem:
There is NO molecule secreted by the organizer and received by the ectoderm
How is neural tissue made from ectoderm?
Organizer – ectoderm – neural tissue
NO!
It is the epidermis induced to form and NOT the neural tissue
Ectoderm is induced to become epidermal tissue by binding to
Bone Morphogenetic Proteins (BMPs)

52. Induction of Neural Ectoderm and Dorsal Mesoderm: BMP Inhibitors
Nervous system forms from region of ectoderm that is protected from epidermal induction (BMP)
By BMP-inhibiting molecules
The “default fate” of ectoderm is to become neural tissue
Certain parts of embryo induce the ectoderm to become epidermal tissue by secreting BMPs
The organizer tissue acts by secreting molecules that block BMPs – thus allowing ectoderm “protected” by BMP inhibitors to become neural tissue

53. Regional Specificity of Neural Induction
Regional differences
Regional specificity of neural structures
Forebrain
Hindbrain
Spinocaudal region
Organizers not only form neural tissue but also specify regions
Can be induced

54. Regional Specificity of Neural Induction
Figure Regional and temporal specificity of induction

55. Specify Left Right Axis
Most parts symmetrical
Heart and internal organs are not
Xenopus gene Xnr1
Xenopus nodal related 1
Expression of nodal gene in the lateral plate mesoderm on the left side of embryo
Determine heart and gut side
Clockwise rotation of cilia keeps it to the left side
Pitx2 activated by Xnr1
Normally expressed on left side and controls which side heart and gut folds occur
7.36 Pitx2 determines the direction of heart looping and gut coiling