Transcript The Organizer - bthsresearch

Xenopus Cleavage and
Gastrulation II:
A Molecular Focus
Gilbert - Chapter 10
Today’s Goals
• Become familiar with the concepts of
Cleavage, Gastrulation and Axis
Determination
• Become familiar with the types of cell
movements in the embryo
• Describe the processes of Cleavage and
Gastrulation in Sea Urchin and Xenopus
embryos
• Become familiar with the types of genes that
help guide gastrulation
As we move on, it will be
important to remember
• Differentiation: the development of
specialized cell types
• Commitment
– Before the cell actually overtly differentiates, a
period of cellular commitment occurs
– Specification
• Reversible
• Autonomous and Conditional
– Determination
• Not reversible
• Mosaic vs. Regulative Development
Amphibian Gastrulation
• We’ll more closely examine some of
the “regulative” aspects of Xenopus
(and newt) gastrulation
• Specifically
– How cells interact with one another during
cell migration
– How cells signal to each other to determine
cell fates
• One more important concept before we
begin . . .
Cell Signaling
• One group of cells changes the
behavior of an adjacent group of cells
– (shape, mitotic rate, fate, gene expression)
• This is called induction
– The cells that will produce signal = inducer
– Cells that receive signal = responder
Cell Signaling
• For this to happen:
– Inducer must produce signal molecule
– Responder must be competent to receive
that signal! (and process it)
– Example:
• Signaling molecule is a secreted growth factor
• Responder must have receptors on the cell
membrane specific to that growth factor to
receive that signal
•What if we waited until the next cleavage to transplant the
cells?
•Would back cells still be competent to receive the signal
that they are now belly tissue?
•Would the belly cells still be secreting that signal?
• So - now back to Amphibian gastrulation
• Let’s apply these concepts. . .
Amphibian Axis Formation
and “The Organizer”
• Amphibian gastrulation and axis
formation are an example of regulative
development
• Inductive interactions occur between
cells
• This was demonstrated by Hans
Spemann and Hilde Mangold
(University of Frieburg, early 1900’s)
– Nobel Prize winners
The Grey Crescent
• If one blastomere does not contain a portion
of the grey crescent, it will not form a normal
embryo
– Conclusion: grey crescent is essential for proper
embryonic development
• What is so special about the grey crescent?
– Fate maps show that these cells form the Dorsal
lip of the blastopore!
– Dorsal lip cells initiate gastrulation
– These cells must be committed when the grey
crescent forms - but how??
– What is in the grey crescent that commits them?
Spemann and Mangold
• Performed many types of transplants at
the early gastrula and late gastrula
stages in the newt embryo
• High amount of technical difficulty!
• Results were fascinating and sent many
developmental biologists on a hunt for
signaling molecules
• These experiments showed that in most
cases, the cells of the embryo are not
committed until at least the late gastrula
stage
• But - There is ONE tissue from the early
gastrula that is already committed. . .
The Organizer
• Spemann dubbed the Dorsal lip of the
blastopore as “the organizer”
– Induced ventral cells to form neural tube
and somites
– Organized the axis of the embryo
The organizer: more questions
than answers!
• How did the organizer get its abilities?
• Why is the dorsal blastopore lip different
than rest of embryo?
• What factors are secreted to cause the
induction of the axes?
The Nieuwkoop Center
• Pieter Nieuwkoop’s and Osamu Nakamura’s
experiments of the 1970’s
• Recombined parts of the blastula to examine
cellular inductions
• They discovered that the dorsal-most vegetal
cells are capable of inducing the dorsal
blastopore lip to begin
– Dubbed these cells the “Nieuwkoop Center”
– Remember: this area is formed by cytoplasmic
rotation after fertilization!
Other findings by Niewkoop
• Found that the vegetal cells that are
destined to become endoderm
– Can induce the ectoderm cells above to
become mesoderm
The Organizer: Molecular
mechanisms
• The discovery of the Niewkoop center’s
induction of the ectoderm (by endoderm) to
form mesoderm sent scientists on a hunt for
the signaling molecule/s involved . . .
– Induction of mesoderm?
– Induction of the organizer?
– How does the organizer induce the series of
events that lead to gastrulation and axis
formation???
Hunting for Signaling
Molecules
• Needed to be able to screen cDNA libraries,
clone the mRNA’s that could mimic these
inductions
• Choosing candidate molecules also became
easier with help from Drosophila studies
– Weischaus, Nusslein-Volhard, Lewis
– Massive mutagenesis screen to find all genes
essential for fly embryogenesis
– Frog embryologists could try out some of those
candidate molecules as well
Niewkoop Molecules
• Xenopus brachyury (Xbra)
– A transcription factor that helps trigger
endoderm induction of the ectoderm to
form mesoderm
– Expressed throughout the vegetal
hemisphere
– NOT a good candidate for induction of the
organizer
Inducing the Organizer
• -catenin
– Protein that accumulates in the dorsal
portion of the egg after fertilization
– 2 known functions: Cell adhesion, nuclear
transcription factor in the WNT signaling
pathway
– A possible candidate
Adherens junction: ß- catenin
ß-catenin and organizer
induction: The evidence
• ß-catenin continues to accumulate in the dorsal
most vegetal cells
– SO: it’s in the right place at the right time! (expressed)
– BUT: can it do the job?? (Overexpression?)
– AND: Is it essential for getting the job done? (KO?)
• Injection of ß-catenin on the ventral side of the
embryo induces a secondary axis
– SO: it can do the job!
• Depletion of ß-catenin using anti-sense
oligonucleotides results in the lack of dorsal
structures
– SO: it is essential for getting the job done!
How does ß-catenin become localized
to the Niewkoop center?
• ß-catenin is initially synthesized from
maternal mRNA’s throughout the embryo
• In the ventral cells, GSK-3 degrades the ßcatenin
• It is proposed that during cytoplasmic
movements after sperm entry, an inhibitor of
GSK-3 is specifically placed at the site
opposite that of sperm entry (future
organizer!)