V. Organizing Power and Axis Formation

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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

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• 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

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Remember our primary axes....
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Figure 5.8 Fate map and cell lineage of the sea
urchin Strongylocentrotus purpuratus
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Step 1 Specification of Micromeres
Two Big Changes Specified to become skeletogenic
mesenchyme
Specified to become Organizer for other cells
egg
disheveled expression blocks B-catenin degradation
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?-catenins job
NML
All endo and meso
ALL
All ecto
NONE
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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

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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

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Spiral cleavage in molluscs
The spirally cleaving mollusks have a strong
autonomous specification from cytoplasmic
determinants in egg.
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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.
TFs associated with the lobe turn CD into The
Organizer
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Figure 5.27 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
The Organizer induces mesodermal and
endodermal fates in cells that would otherwise
remain ectodermal
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MAP kinase activity activated by D-quadrant snail
blastomeres
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Figure 5.30 MAP kinase activity activated by
D-quadrant snail blastomeres (Part 2)
Normal
MAPK Blocked
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Axis Determination

• Anterior-Posterior Cytoplasmic determinants in
  the lobe
• Left-Right Nodal expression (TGF-B family
  member)
• Dorsal-Ventral Cytoplasmic determinants in the
  lobe

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Bilateral, Holoblastic Cleavage of the Tunicate
The 8-cell embryo is already autonomously specifie
d for cell fates
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Figure 5.35 Cytoplasmic rearrangement in the
fertilized egg of Styela partita
Fertilization rearranges cytoplasmic determinants
1. Animal pole cytosol determines ectoderm 2.
B-catenin presence determines endoderm (like
urchins) 3. Macho-1 in yellow crescent determines
muscle cells
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Figure 5.38 Antibody staining of ?-catenin
protein shows its involvement with endoderm
formation
Wherever B-catenin shows up, endoderm is formed
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Figure 5.37 Autonomous specification by a
morphogenetic factor
Where Macho-1 shows up tail muscle will form
Zinc-finger TF for muscle actin, myosin,
TBX-6 Also TF for Snail TF which blocks notochord
induction
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Conditional Specification also plays a role
Integrates with the autonomous specification
patterns
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Axis Formation accomplished prior to cleavage!
Fertilization rearranges cytoplasmic determinants
determines dorsal-ventral
determines anterior-posterior
Left-right unclear but nodal shows it later
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Rotational, Holoblastic Cleavage in the nematode
Caenorhabditis elegans
hermaphrodite
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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
Founder cell divisions are equatorial
AB requires input from P lineage
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• 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

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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)
Sperm CYK-4 activates egg rho, actin
rearrangement causes assymetric first cleavage
division
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AB division leads to both dorsal ventral and
left-right axes
Assymetrical division of AB-MS forces AB dorsal
and MS ventral
Delta-notch recognition between daughters of AB
and MS gives left-right
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Cytoskeletal rearrangement also pushes
P-granules into the germ line
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The cells of the blastula have specified fates in
Xenopus.....
Gastrulation changes all of that,
.....afterwards all cell fates are determined!
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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.
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The dorsal mesoderm keeps the power to determine
other cells fates throughout gastrulation
Spemanns Organizer
This ability to determine cell fates is
called... Primary Embryonic Induction
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The dorsal lip cells first have to become
competent to be Organizer
Cortical rotation shifts disheveled, GBP,
Wnt-11 to dorsal side of embryo
The area of Dsh accumulation is seen as a
gray crescent in some amphibian embryos
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ß-catenin starts out everywhere in the embryo but
only survives GSK3 in the dorsal portion due to
Dsh, GBP and Wnt-11
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The dorsal vegetal cells of the Nieuwkoop Center
turn on Organizer
Wnt and Vg-1 (TGF-B family) induce pre-dorsal lip
mesoderm
FGF needed for all mesoderm
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Figure 7.22 Summary of events hypothesized to
bring about induction of the organizer in the
dorsal mesoderm
Nodal
Vg-1
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Figure 7.23 Vegetal induction of mesoderm (Part
2)
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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

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Figure 7.26 Localization of chordin mRNA
The Organizer is induced prior to gastrulation
Dorsal blastopore lip
Blastopore
Dorsal mesoderm
Continues to organize events throughout its own
differentiation
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Interestingly, the primary mechanism is by means
of inhibition....
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Presumably, the Wnt, FGF and RA signals arise
from endoderm and ectoderm
Without the Organizer you get mainly skin and
gut
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Figure 7.31 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
Dont underestimate the power of the Organizer!
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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

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Nodal expression causes Pitx2 expression
Nodal and Pitx2 on left
Injected on both sides
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Relationships between the frog and chick
Organizers

• The hypoblast dorsal vegetal cells
• Kollers sickle pre-dorsal lip mesoderm
• Hensens node dorsal blastopore lip and dorsal
  mesoderm
• Primitive streak blastopore

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Formation of Hensens node from Kollers sickle
Wnt and FGF from the hypoblast induce Kollers
sickle epiblast
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Figure 8.10 Induction of a new embryo by
transplantation of Hensens node (Part 1)
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Possible contribution of inhibition of BMP
signaling
Appears to be similar to the frog....
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In the chick, the hypoblast plays a large role
much like the frog endoderm
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Figure 8.8 Specification of the chick
anterior-posterior axis by gravity
Anterior-Posterior axis parallels the rotation
inside the shell
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Rostral-Caudal (Anterior-Posterior) axis
extension in chick embryos
The combination of positional specification, compl
ex signaling and TF (Hox, etc.) expression is
thought to cause axis.
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Left-right asymmetry in the chick embryo
This is farther along Nodal and
Pitx2 again are implicated