Download Mesoderm and Endoderm Formation

“It is not birth, marriage or death,
but gastrulation which is truly the
most important time in your life.”
Lewis Wolpert
Gastrulation
1. Mesodermal and Endodermal Cell Movements
(i.e. Establishment of Cell Fate)
2. Mesoderm and Endoderm Formation
3. Establishment or Molecular Manifestation of
Anterior-Posterior and Doral-Ventral Axes
4. Right-Left Symmetry
5. Growth
Gastrulation
1. Mesodermal and Endodermal Cell Movements
(i.e. Establishment of Cell Fate)
2. Mesoderm and Endoderm Formation
3. Establishment or Molecular Manifestation of
Anterior-Posterior and Doral-Ventral Axes
4. Right-Left Symmetry
5. Growth
Vertebrate
Embryonic Lineage
Mesoderm and Endoderm
Formation (Induction)
• Mesoderm and Endoderm Induction.
– Definitions and properties
– Nieuwkoop’s Studies
• Using Nieuwkoop’s Studies to Indetify
Candidate Molecules and Molecular Pathways
of Mesoderm Formation.
– TGF-ßs
– FGFs
• Present State of Affairs.
The 3 Germ Layers
• Ectoderm, e.g. epidermis, neural plate,
neural crest.
• Mesoderm, e.g. notochord, muscle,
kidney, blood, heart
• Endoderm, e.g. pharynx, liver, stomach,
pancreas
Nieuwkoop’s Classic Induction
Experiments
Conclusions from
Nieuwkoop’s Experiments
1. Mesoderm is induced when ectoderm and
endoderm are juxtaposed.
Repeat But With Labeled Ectoderm and
Endoderm (i.e. lineage tracers)
Conclusions from
Nieuwkoop’s Experiments
1. Mesoderm is induced when endoderm and
ectoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm; i.e. the ectoderm is induced to form
mesoderm.
INDUCTION
Process by which one group of cells
signals to a second group of cells
altering their ultimate developmental
state
The Two Primary Components of
an Inductive System
1. A signal generated by the inducing
cell.
2. A receptive system that directly or
indirectly controls gene expression
in the responding cell, i.e.
competence.
Repeat with filter or defined distance
between ectoderm and endoderm
Conclusions from
Nieuwkoop’s Experiments
1. Mesoderm is induced when endoderm and
endoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm.
3. The signal from the endoderm is secreted.
Post- Nieuwkoop’s
Experiments
1. How is the “Signal” propagated?
2. How far is the signal propagated?
Conclusions from
Nieuwkoop’s Experiments
1. Mesoderm is induced when endoderm and
endoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm.
3. The signal from the endoderm is secreted.
4. The signal can only act over a short distance
(4-5 cell diameter).
How?
The Diffusion verses Relay Debate
Diffusion
Relay
When is the signal present?
COMPETENCE
Ability of a group of cells to
respond to an inducing signal.
The competence is directly
related to the age of a tissue.
Repeat Varying Age of ‘Donor’
(Endoderm) and ‘Recipient’ (Ectoderm)
Further Conclusions from
Nieuwkoop’s Experiments
2 hours some induction of muscle
5 hours complete induction of muscle
11 hours no induction can occur
i.e. Formative Effect (length of time not dose of
inducer)
Mesodermal competence is not associated with
protein synthesis and occurs in a cell autonomous
manner,i.e. internal clock.
Mesoderm and Endoderm
Induction
• Mesoderm and Endoderm Induction.
– Definitions and properties
– Nieuwkoop’s Studies
• Candidate Molecules and Molecular Pathways
of Mesoderm Formation.
– TGF-ßs
– FGFs
• Present State of Affairs.
What is the endogenous
signal(s)?
Methods to Identify the Endogenous
Mesoderm Inducing Signal
1. Cut and Dump: Place animal cap tissue in the
presence of potential mesodermal inducer-score for
expression of mesodermal markers and cellular
movements.
2. Functional Screen: Inject embryos with potential
gene or cDNA library, cut animal caps score as
above.
3. Expression Screen:
a. Differential screen between ectoderm and
endoderm. Alternatively b. Pick cDNA from tissue
specific library and perform in situs.
Methods to Identify the
Endogenous Mesoderm Inducing
Signal
1. Cut and Dump: Place animal cap tissue in
the presence of potential mesodermal
inducer.
Animal Cap Assays
Methods to Identify the
Endogenous Mesoderm Inducing
Signal
2. Functional Screen: Inject embryos with
potential gene or cDNA library, cut animal
caps score as above.
Brachyury
Brachyury is Required for Posterior
Mesoderm Formation
Mouse Brachyury Expression
Xbra Expression
T-Box Proteins
• Family of classical transcription factors that share
sequence homology within DNA binding domain (T-box).
• Constitute approximately 0.1% of all genomes
• Function as ‘Classical Transcription Factors’
– Localize to the nucleus
– Bind DNA in a sequence specific fashion
– Regulate transcription of neighboring genes
• Multiple family members are expressed in specific
domains within the developing organs and tissues
• Mutated in Development and Disease
Methods to Identify the
Endogenous Mesoderm Inducing
Signal
3. Expression Screen:
a. Differential screen between ectoderm and
endoderm.
b. Pick cDNA from tissue specific library and
perform in situs.
Identify Endoderm Specific
Molecules
Subtractive Hybridization
e.g. Veg
Criteria for Mesodermal Inducer Based on
Nieuwkoop’s Studies
1. Gene or protein must be expressed and active in the
correct time and place.
2. Protein must be secreted and act at a distance.
3. Protein must be necessary and sufficient to induce
mesoderm in ectoderm in a protein synthesis
independent fashion.
Every molecule ever identified in 30+
years of screening that fits this
criteria is either:
1. A member of the TGF-ßs superfamily
2. A member of the FGF superfamily
Is the Endogenous Signal
BMP?
Conclusions from
Nieuwkoop’s Experiments
1. Mesoderm is induced when endoderm and
endoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm.
3. The signal from the endoderm is secreted.
4. Mesoderm induction is independent of
protein synthesis.
5. The signal can only act over a short distance
(4-5 cell diameter).
Problems with
BMP/Activin/Nodal
1. Mesoderm is induced when endoderm and
endoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm.
3. The signal from the endoderm is secreted.
4. Mesoderm induction is independent of
protein synthesis.
5. The signal can only act over a short distance
(4-5 cell diameter).
Every molecule ever identified in 30+
years of screening that fits this
criteria is either:
1. A member of the TGF-ßs superfamily
2. A member of the FGF superfamily
Animal Cap Assays
FGF Signaling
FGF is Required for Formation
of Posterior and Ventral
Mesoderm
Is FGF an Endogenous Inducing
Signal?
Conclusions from
Nieuwkoop’s Experiments
1. Mesoderm is induced when endoderm and
endoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm.
3. The signal from the endoderm is secreted.
4. Mesoderm induction is independent of
protein synthesis.
5. The signal can only act over a short distance
(4-5 cell diameter).
Problems with FGF
1. In regards to mesoderm formation or
patterning, no gene has yet to be identified
that is solely FGF inducible.
2. FGF can only induce posterior mesoderm
Animal Cap Assays
FGF Signaling
Problems with FGF
1. Mesoderm is induced when endoderm and
endoderm are juxtaposed.
2. Signals arise from endoderm and received by
ectoderm.
3. The signal from the endoderm is secreted.
4. Mesoderm induction is independent of
protein synthesis.
5. The signal can only act over a short distance
(4-5 cell diameter).
Is FGF AND BMP the
Endogenous Inducing Signal?
Is it something altogether
different?