1. V. Organizing Power and Axis Formation
Background Information
B. Invertebrates
Sea Urchins
Snails
Tunicates
C. Elegans
Drosophila melanogaster
C. Vertebrates
The Frog
Zebrafish
The Chick Embryo
Mammals

2. Part of these processes is the determination of axes in the organism
The first few cleavages may produce little or no directionality to the embryo
It starts at varying stages in various animals and can result from different mechanisms

3. Remember our primary axes....
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4. Figure 5.8 Fate map and cell lineage of the sea urchin Strongylocentrotus purpuratus
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5. Step 1: Specification of Micromeres
Two Big Changes: Specified to become skeletogenic mesenchyme
Specified to become “Organizer” for other cells
egg
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disheveled expression blocks B-catenin degradation

6. -catenin’s job NML All endo and meso ALL All ecto NONE
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All
ecto
NONE

7. Step 2: “Organizing Power”
Secrete Wnt-8 into autocrine loop
Wnt-8Blimp-1B-cateninWnt-8
Paracrine “early signal” induces macromeres and vegetal cells to differentiate to vegetal endoderm
Unknown signal as of yet
Delta-Notch juxtacrine signal induces non-skeletogenic mesenchyme
Wnt-8 makes a come-back to induce invagination

8. Axis Determination
Anterior-Posterior: Cytoplasmic determinants in the egg cytosol, such as disheveled and B-catenin
Left-Right: Nodal expression (TGF-B family member)
Dorsal-Ventral: unclear

9. Spiral cleavage in molluscs
The spirally cleaving mollusks have
a strong autonomous specification
from cytoplasmic determinants in egg.
DevBio9e-Fig jpg

10. Step 1: Polar lobe formation
The polar lobe
is a cytoplasm
outpouching
from the egg
prior to cleavage
It isolates critical
determinants into
only one of the
first cell pair.
TF’s associated
with the lobe
turn CD into
“The Organizer”
DevBio9e-Fig jpg

11. Decapentaplegic is TGF-B family member used to
Figure Association of decapentaplegic (dpp) mRNA with specific centrosomes of Ilyanassa
Decapentaplegic is TGF-B family member used to
induce specific cell fates secreted by the Organizer
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The Organizer induces mesodermal and endodermal
fates in cells that would otherwise remain ectodermal

12. MAP kinase activity activated by D-quadrant snail blastomeres
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13. Figure 5.30 MAP kinase activity activated by D-quadrant snail blastomeres (Part 2)
Normal
MAPK Blocked
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14. Axis Determination
Anterior-Posterior: Cytoplasmic determinants in the lobe
Left-Right: Nodal expression (TGF-B family member)
Dorsal-Ventral: Cytoplasmic determinants in the lobe

15. Bilateral, Holoblastic Cleavage of the Tunicate
The 8-cell embryo is
already autonomously
specified for cell fates
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16. Fertilization rearranges cytoplasmic determinants
Figure Cytoplasmic rearrangement in the fertilized egg of Styela partita
Fertilization rearranges cytoplasmic determinants
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1. Animal pole cytosol determines ectoderm
2. B-catenin presence determines endoderm (like urchins)
3. Macho-1 in yellow crescent determines muscle cells

17. Wherever B-catenin shows up, endoderm is formed
Figure Antibody staining of -catenin protein shows its involvement with endoderm formation
Wherever B-catenin shows up, endoderm is formed
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18. Figure 5.37 Autonomous specification by a morphogenetic factor
Where Macho-1 shows up tail muscle will form
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Zinc-finger TF for muscle actin, myosin, TBX-6
Also TF for Snail TF which blocks notochord induction

19. Conditional Specification also plays a role
Integrates with the autonomous specification patterns
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20. Axis Formation accomplished prior to cleavage!
Fertilization rearranges cytoplasmic determinants
determines
dorsal-ventral
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determines
anterior-posterior
Left-right: unclear
but nodal shows it later

21. Rotational, Holoblastic Cleavage in the nematode Caenorhabditis elegans
DevBio9e-Fig R.jpg
hermaphrodite

22. Figure 5.42 The nematode Caenorhabditis elegans (Part 2)
Both autonomous and conditional
specification at work early on.
If cells are separated:
P1 will develop autonomously
Stem cell divisions are meridional
DevBio9e-Fig R.jpg
Founder cell divisions are equatorial
AB requires input from P lineage

23. Autonomous specification in P1
SKN-1, PAL-1 and PIE-1 TFs from egg
as P1 divides these determine daughter fates
P lineage becomes “Organizer”
Conditional specification in AB
P2 secretes Wnt family member MOM-1 to induce endodermal specification in AB lineage
P2 use Delta-Notch signals to induce ectodermal fates in AB lineage

24. Axis Determination in C. elegans
Anterior-Posterior axis
is determined by egg shape
Which end is posterior
is determined by sperm
(the closest end is back)
DevBio9e-Fig jpg
Sperm CYK-4
activates egg rho,
actin rearrangement
causes assymetric
first cleavage division

25. AB division leads to both dorsal ventral and left-right axes
Assymetrical division of AB-MS forces AB dorsal and MS ventral
DevBio9e-Fig jpg
Delta-notch recognition between daughters of AB and MS gives left-right

26. Cytoskeletal rearrangement also pushes P-granules into the germ line
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27. The cells of the blastula have specified fates in Xenopus.....
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Gastrulation changes all of that,
.....afterwards all cell fates are determined!

28. Development of “Organizing Power” at the dorsal blastopore lip
The bottle cells
get the ball rolling
but the real power
is conferred on the
first cells through
the blastopore.
DevBio9e-Fig R.jpg

29. This ability to determine cell fates is called...
The dorsal mesoderm keeps the power to determine other cell’s fates throughout gastrulation: “Spemann’s Organizer”
This ability to determine
cell fates is called...
“Primary Embryonic Induction”
DevBio9e-Fig R.jpg

30. The dorsal lip cells first have to become competent to be “Organizer”
Cortical rotation shifts
disheveled, GBP, Wnt-11
to dorsal side of embryo
DevBio9e-Fig R.jpg
The area of Dsh
accumulation is
seen as a gray
crescent in some
amphibian embryos

31. β-catenin starts out everywhere in the embryo but only survives GSK3 in the dorsal portion due to Dsh, GBP and Wnt-11
DevBio9e-Fig R.jpg

32. The dorsal vegetal cells of the Nieuwkoop Center turn on “Organizer”
Wnt and Vg-1 (TGF-B family)
induce pre-dorsal lip mesoderm
DevBio9e-Fig R.jpg
FGF needed for all mesoderm

33. Figure Summary of events hypothesized to bring about induction of the organizer in the dorsal mesoderm
Nodal
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Vg-1

34. Figure 7.23 Vegetal induction of mesoderm (Part 2)
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35. So, what can the “Organizer” do?
Initiate gastrulation
Become the notochord and other dorsal mesoderm
Dorsalize ventral mesoderm into paraxial mesoderm, somites, etc.
Dorsalize the ectoderm into the neural plate and neural tube

36. DevBio9e-Table jpg

37. Figure 7.26 Localization of chordin mRNA
The “Organizer” is induced prior to gastrulation
Dorsal blastopore lip
Blastopore
Dorsal mesoderm
DevBio9e-Fig jpg
Continues to organize events throughout its own differentiation

38. Interestingly, the primary mechanism is by means of inhibition....
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39. Without the “Organizer” you get mainly skin and gut
Presumably, the Wnt, FGF and RA signals arise from endoderm and ectoderm
Without the “Organizer”
you get mainly skin and gut
DevBio9e-Fig jpg

40. Don’t underestimate the power of the “Organizer”!
Figure Cerberus mRNA injected into a single D4 blastomere of a 32-cell Xenopus embryo induces head structures as well as a duplicated heart and liver
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Don’t underestimate the power of the “Organizer”!

41. Axis Formation
Dorsal-Ventral: sperm penetration and cortical rotation
Anterior-Posterior: migration direction of the dorsal mesoderm
Left-Right: nodal expression exclusively on left side of the lateral plate mesoderm

42. Nodal expression causes Pitx2 expression
Nodal and Pitx2 on left
Injected on both sides
DevBio9e-Fig jpg

43. Relationships between the frog and chick “Organizers”
The hypoblast = dorsal vegetal cells
Koller’s sickle = pre-dorsal lip mesoderm
Hensen’s node = dorsal blastopore lip and dorsal mesoderm
Primitive streak = blastopore

44. Formation of Hensen’s node from Koller’s sickle
DevBio9e-Fig jpg
Wnt and FGF from the hypoblast
induce Koller’s sickle epiblast

45. Figure 8.10 Induction of a new embryo by transplantation of Hensen’s node (Part 1)
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46. Possible contribution of inhibition of BMP signaling
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Appears to be similar to the frog....

47. In the chick, the hypoblast plays a large role much like the frog endoderm
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48. Anterior-Posterior axis parallels the rotation inside the shell
Figure 8.8 Specification of the chick anterior-posterior axis by gravity
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Anterior-Posterior axis parallels the rotation inside the shell

49. Rostral-Caudal (Anterior-Posterior) axis extension in chick embryos
The combination of
positional specification,
complex signaling and
TF (Hox, etc.) expression
is thought to cause axis.
DevBio9e-Fig jpg

50. Left-right asymmetry in the chick embryo
This is farther along
Nodal and Pitx2 again are implicated
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