chapter 10, early development and axis formation in amphibians part-2

CHAPTER 10, EARLY
DEVELOPMENT AND AXIS
FORMATION IN AMPHIBIANS
PART-2
ZOO3603C

Progressive Determination of the
Amphibian Axes

The specification of axes
Vegetal proteins
VegT destroyed--- entire embryo develops as an
epidermis
Vg1 lacked --- no endoderm nor dorsal mesoderm
The axes are specified by events triggered at
fertilization and realized during gastrulation.

Concept of regulative development
Blastomere has a potency greater than its normal
embryonic fate.
Fate is determined (induced) by interactions
between neighboring cells.
How is it induced
What factor cause the induction

Spemann’s demonstration of nuclear
equivalence in newt cleavage
Ligature at 8-cell stage
Entering a single nucleus
to the other side at 16-
cell stage
Nuclei at 16-cell stage are genetically identical
and still totipotent in the newt.
After 14 days, each side had
a normal embryo.

Asymmetry in the amphibian egg
Separation at
left-right axis
Separation at
ventral-dorsal axis
Containing
epidermal, blood,
mesenchymal and
gut cells.
Gray crescent is
essential for
normal
development.

Determination of ectoderm during newt
gastrulation
Normal development
Regulative development
What does cause the determination
Secondary neural plate
Autonomous development

Hans Spemann and Hilde Mangold:
Primary Embryonic Induction

Organization of a secondary axis by
dorsal blastopore lip tissue
Dorsal blastopore is the
self-differentiating
tissue.
• Organizer
• Primary embryonic induction

Mechanisms of Axis Determination in
Amphibians
• How does the organizer form
• The dorsal signal: -catenin
• The vegetal TGF--like signal
• The mesodermal signal

Questions on the mechanisms
How did the organizer get its properties
What caused the dorsal blastopore lip to differ from
any other region of the embryo
What factors were being secreted from the
organizer to cause the formation of the neural tube
and to create the anterior-posterior, dorsal-ventral
and left-right axes

Mesodermal induction by vegetal endoderm
Animal cap cells generate
mesodermal tissue.
Factors from vegetal cells

Mesodermal induction by vegetal endoderm
On dorsal side, a signal is
released by Nieuwkoop center.

Signals for the axial determination
The dorsal signal: -catenin
The vegetal TGF--like signal
The mesodermal signal, Nodal-relating proteins

The vegetal cells are responsible for causing
the initiation of gastrulation
Rescue by transplantation of dorsal vegetal blastomeres which
can induce another axis in the embryo.
Vegetal cells induce the
organizer which induces axis.

The regional specificity of mesoderm induction
D1 most
induced dorsal
mesoderm.
Nieuwkoop Center
is in D1 cell
What makes
dorsal/ventral
mesoderm

The role of Wnt pathway in ventral-dorsal
axis specification
2-Cell
-catenin
Blastula
-catenin localize in nuclei on
dorsal, but not on ventral.
Dorsal
Ventral
-catenin dorsal localization
persists through gastrula stage.
How this localization happen

Model of the mechanism of localization of
-catenin in the dorsal portion
Dsh, Dishevelled protein
GBP, GSK3-binding protein
GSK3, glycogen synthase kinase 3
on microtubules
cortical rotation
Cortical rotation causes the sift
of the Dsh complex.

Model of the mechanism of localization of
-catenin in the dorsal portion
1. Dsh inhibits GSK3
2. GSK3 degrade -catenin
3. -catenin initiate the organizer
Dsh localization cause the ventraldorsal
axis

Model of the mechanism of localization of
-catenin in the dorsal portion
Inactivation of GSK3 on both
blastomeres of 2 cell.
Formation of 2 nd axis.
GSK3 is one of the key to
determine the ventral-dorsal axis
through -catenin.

Induction of the organizer in the dorsal
mesoderm
• Tcf3, ubiquitous transcription factor
• -catenin/Tcf3 complex can
activate the transcription.
• Goosecoid protein, transcription
factor which can activate genes in
organizer.
Difference of -catenin
expression cause the ventraldorsal
axis.

Mesoderm induction and organizer formation
by -catenin and TGF- proteins
Xnr, nodal-related gene
induced by -catenin,
Veg1 and Vg1.
Goosecoid induction

Functions of the Organizer
• Induction of neural ectoderm and dorsal
mesoderm: BMP inhibitors
• Noggin
• Chordin
• Follistatin

Four major ability of the organizer
Self-differentiate dorsal mesoderm.
Dorsalize the surrounding mesoderm into paraxial
mesoderm.
Dorsalize the ectoderm, including the formation of
neural tube.
Initiate the movements of gastrulation.

Ability of goosecoid mRNA to induce a
new axis
Gastrula
Control Injected
goosecoid mRNA
+goosecoid
Single
blastopore
Double
blastopore
Control
Two dorsal axes Two heads
Goosecoid can induce the axis.

Neural structures induced in presumptive
ectoderm
Dorsal
lip
Ectoderm
Filter membrane
Incubation of ectoderm with dorsal lip
without the direct contacts induced
neural structures.
Secreted factors from dorsal
lip induced the neural
differentiation.
What is the secreted factors
from dorsal lip

Rescue of dorsal structures by Noggin
protein
Control
Exposure to UV causes no the
cortical rotation.
No organizer
+ noggin mRNA
Noggin mRNA injection rescue the
dorsal structure in dosagerelated
fashion

Localization of noggin mRNA in the
organizer tissue
In situ Hybridization
Gastrulation
Involution
Blastopore lip
Convergent
extension
Prechordal plate
& pharyngeal
endoderm
Extend
beneath the
ectoderm

Localization of chordin mRNA
In situ Hybridization
Just prior to
gastrulation
Chordin, one of the organizer protein
Beginning gastrulation,
Dorsal blastopore lip
Later gastrulation,
organizer tissues
Chordin expression is activated by -catenin

Model for the action of the organizer
Organizer molecules block
the action of BMP4.
Immuno-histo chemistry of Smad1 which is
downstream of the cascade BMP4.
Organizer molecules induce ventral-dorsal
axis through BMP4 signaling pass way.

Control of neural specification by the
levels of BMPs
Whole mount in situ hybridization of Sox2, which is a specific mRNA in
neural tube
Control
Hyper BMPs by morpholino.
Lack of Sox2 expression in
neural tube
Control
Hypo BMPs
Epidermis is instructed by BMP
signaling, and the organizer works by
blocking the BMP signal from reaching
the ectoderm above it.
Lack of ventral-dorsal axis in
neural tube

The regional Specificity of Induction
• The determination of regional differences
• The head inducer: Wnt inhibitors
• Cerberus
• Frzb and Dickkopf
• Insulin-like growth factors
• Trunk induction: Wnt signals and retinoic acid

Regional specificity of induction
Transplantation of archenteron roof into blastocoel in early gastrulae.
Head with balancers
Head with balancers,
eyes and forebrain
Posterior part of head,
diencephalon and otic
vesicles
Trunk-tail segment

Regionally specific inducing action of the dorsal
blastopore lip
Early blastula dorsal lips
induce anterior dorsal
structures
Older blastula dorsal lips
induce more posterior
dorsal structures
Earlier organizer: induce brains and heads
Later organizer: induce spinal cords and tails
Region and timing makes difference.

Paracrine factor antagonists from the
organizer
What does induce the head Wnt inhibitors
Secrete from
Both active
Both inactive
Epidermis is formed by
having both the Wnt and
BMP signaling pathways.

Cerberus induces head structures as well as a
duplicated heart and liver
Injection of Cerberus mRNA into D4 cell (ventral;
vegetal) at 32-cell stage.
Head structures, heart and liver were induced.
By blocking BMPs, Nodalrelated
protein and Wxnt8,
cerberus can induce a head.

Xwnt8 is ventralizing the mesoderm and
preventing anterior head formation
• Frzb and Dickkopf protein is
secreted by the anterior region of
the organizer.
• Frzb and Dickkopf bind to Xwnt8.
• Block the ventralizing function of
Xwnt8.

Insulin-like growth factors enhance anterior
neural development
Expression of
Igf3 in anterior
neural tube
An ectopic headlike
structure
induced by Igf2
mRNA injection.
IGF inhibition
The cement gland and
eyes are absent.
Control
+IGF receptor inhibitor
at 4-cell stage

Trunk induction: The Wnt signaling pathway
and posteriorization of the neural tube
-catenin density in future neural plate
Changing the level of Wnt signaling alters the expression of
regionally specific markers in neurula.
Control +Xwnt8 +Xfrzb1
Less anterior
More anterior
Bf1, forebrain; Otx2, forebrain & midbrain; Krox20, two region of hindbrain

Trunk induction: The Wnt signaling pathway
and posteriorization of the neural tube
Double-gradient model
o BMP gradient: ventral-dorsal axis
Ventral > Dorsal
o Wnt gradient: anterior-posterior axis
Anterior < Posterior on neural plate

Model of organizer function and axis
specification in the Xenopus gastrula
Anteriorposterior
axis
Ventraldorsal
axis
IGFs

Specifying the left-right axis

Left-Right Axis: Pitx2 determines the
direction of heart looping and gut coiling
Wild-type
Injected Pitx2
Pitx2 in just left
side of mesoderm.
Counter-clockwise
gut coiling
Pitx2 in both side
of mesoderm
Random gut
coiling
Nodal protein establishes left-light polarity by activating Pitx2 on
the left side of embryo.

Next class
Chapter-11: The early development vertebrates:
fish, birds and mammals