ppt slides

MCDB 4650 Class 8
The Nieuwkoop center, the Organizer, and
their inductive properties: molecules involved
Learning goals:
• Describe the factors involved in mesoderm and neural ectoderm
determination
• Relate the concept of morphogens to the determination of mesoderm
and the role of the organizer in Xenopus
• Interpret experiments on this topic and determine possible outcomes.
No problem solving session at 3 pm on Friday: instead, if you have
questions, please go to Roni’s office hour from 2:30-3:30

Scientists had identified three classes of mesoderm:
Dorsal
Intermediate
Ventral
What molecules induce mesoderm?
From previous experiments:
• believed the signal was diffusible and likely to be a
morphogen—higher concentration on dorsal side than
ventral
• believed the signal came from vegetal cells
Find candidate proteins from the vegetal cells

What should the assays be for
discovering mesoderm
inducers?

Simplest:
Look at
phenotype
of cells in
culture
after
treatment

More sophisticated: look at molecular markers
Cardiac actin, only made in heart mesoderm, is thus a marker for this
type of mesoderm

How to start?
Isolate proteins located (or produced) only on the
vegetal side of the embryo
See which of them induce which kinds of
mesoderm.

Candidate molecule: activin (a TGFβ family ligand)
V
Activin?
TGF-b family of molecules
BMP, Nodal, TGF-b, activin
All bind to TGF-b receptor families (dimer of type1
and type 2 subunits)
and activate Smad transcription factors
D

In the experiment below, activin containing beads were placed in the
middle of animal cap cells isolated from a blastula stage embryo.
3.21
Candidate molecule: activin (a TGFβ family ligand)
Brachyury and goosecoid are transcription factors activated by activin

From this data, which cells are likely to express goosecoid, a
transcription factor activated by high concentrations of
activin?
a. Ventral mesoderm
b. Intermediate mesoderm
c. Dorsal mesoderm

Treat isolated animal pole cells with different
concentrations of activin
What does this experiment tell you?
a. Activin is a morphogen
b. Activin can induce mesoderm
c. Activin is required for mesoderm
induction
d. a and b
e. All of the above

Is activin actually responsible for mesoderm induction?
Of the following, which would you choose to
demonstrate that activin was required for
mesoderm induction?
a. Show that activin is expressed in the embryo
at the right time
b. Show that activin is expressed in the embryo
at the right time and in the right place
c. Show that activin can induce mesoderm in
culture
d. Remove activin and observe phenotype
e. Add extra activin and observe phenotype.
Activin does induce mesoderm in vitro….
but so do other molecules that may not actually be responsible for
this fate in vivo

Xenopus nodal related (Xnr), also a TGF-beta ligand is
the main mesoderm inducing signal

Using the kinds of experiments we just discussed, it
has been shown that there is a sequence of 4
signaling events that pattern the early embryo
1. Vegetal pole cells to overlying marginal zone (low levels of low levels
of Xnr)
2. Dorsal vegetal cells (Nieuwkoop center) to overlying cells (future
organizer) (high levels of Xnr)
Up next: steps 3 and 4

Organizer cells make the goosecoid transcription factor
Activated in cells
of organizer by
Xnr signals from
Nieuwkoop center
Smad2/4 transcription factors
Only made in
organizer cells
Goosecoid activates expression of ligands then
secreted from organizer cells

• Put together what you know:

How do the early induction processes fit together?
Concept map the following terms
– Dorsal vegetal cells of the blastula
– Ventral vegetal cells of the blastula
– Nieuwkoop center
– Organizer
– Ventral mesoderm
– Dorsal mesoderm
– Cortical rotation
– Fertilization
– Gastrulation
– Vg1
– Disheveled (Dsh)
– Xnrs
– B-catenin
– Goosecoid
Optional: try linking these terms
together by position in the embryo as
well:
• Draw an embryo, and place the terms
inside the embryo to indicate where
the structures are located, or events
take place
• Label the axes!

Organizer cells are critical for
formation of neural tissue from
the ectoderm
Without the organizer
-no neural ectoderm forms
-no dorsal mesoderm
-other mesodermal fates
missing as well
O

1 st candidate: a gene called Noggin
expressed in dorsal blastopore lip
and notochord
Increased concentrations of
noggin increased the amount
of dorsal structures

Two more groups of researchers were also looking for the
neural inducing factor at the same time (early 1990s)
– Differential screen for
genes activated by
goosecoid pulled out
chordin
– Looking for mesoderm
inducers… found
follistatin, a factor
that inhibited activin
Chordin expression

The 3 candidates (Noggin, Chordin, Follistatin)
are all TGF-beta ligands
So, scientist blocked the TGFb receptor in oocytes
to see what would happen to the animal cap cells
(some of which should make neural ectoderm).

San
This experiment suggests:
es
a. TGFb ligands are required for neural differentiation
b. TGFb ligands are not required for neural differentiation
Mutant TGFb receptor mRNA
(dominant negative)
Neural tissue

Another experiment supported these findings: when animal
cap cells were dissociated in culture, they made neural
tissue on their own
Why?

Remove all BMP from the embryo, and
the embryo makes only neural
ectoderm, no epidermis

normal
BMPs inhibited
with antisense
oligos
Sox-2 expression
(a neural gene)
Chordin, noggin
follistatin
inhibited with
antisense oligos
BMPs and ADMP
inhibited

Which of the following sums up the evidence?
a. BMPs secreted in the late blastula block nonneural
fate
b. Molecules secreted by the organizer in the early
blastula stage are inhibited by BMPs
c. Molecules secreted by the organizer in the late
blastula stage inhibit BMP signaling to allow
neural fate
d. Molecules secreted by organizer induce neural
fate from otherwise non-neural cells

Step 3: secretion of “organizer
molecules” : inhibitors of BMP
Step 4: secretion of BMP from ventral
mesoderm

The default fate
of the ectoderm
is neural
The organizer
induces by
inhibiting an
inhibitor
(BMPs)
Sanes, Reh and Harris, 2000

Animal cap cells not committed at blastula stage, but are by
gastrulation

Summary
• BMPs are present throughout the embryo at the
blastula stage, but downregulated on the dorsal
side by organizer (including by goosecoid)
• The BMP blockers, Noggin (and related molecules),
are secreted before gastrulation, defining neural
ectoderm and intermediate mesoderm.
True in all vertebrates, and many invertebrates as well

What would happen if you removed all
chordin from the Xenopus embryo?
a. no mesoderm could form
b. no neural ectoderm could form
c. no non-neural ectoderm could form
d. no obvious effect

Assume that you can ectopically express
(ectopically=express in a different area) chordin on the
ventral side of a Xenopus gastrula stage embryo. What will
happen?
a. Extra neural structures
b. Extra dorsal mesodermal structures
c. Extra endodermal structures
d. Extra neural and dorsal mesodermal structures
e. Extra neural, dorsal mesodermal and endodermal
structures

Which of the following is NOT true of the cells of
the organizer?
a. Induces gastrulation
b. Inhibits epidermal fate
c. Converts ventral mesoderm to intermediate
mesoderm
d. Involutes through the dorsal lip of the blastopore
e. Becomes intermediate mesoderm—cells that make
up kidney, other organs central to the body