ppt slides

Neurons migrate “inside-out” to form cortical layers:
• deep layer cells are born first
• superficial cells are born later

Bohner, Akers and McConnell 1997
Isolate ventricular
zone cells from early
stage brain (E29)
Label with 3H
Thymidine during
dissociation
Let sit in culture for
6 hours (about the
time observed in vivo
for completion of
final mitosis)
Little purple dots are
3H-T label

Once cells removed, they varied the culture conditions
1. Low density 2. Pellet 3. Explant
No contacts Pellet culturedifferent
contacts
Cells in S phase
Mitotic cells
Normal contacts
After 6 hours in culture, dissociate cells, inject cell (transplant)
into P1 (late stage) ventricular zone.
What’s your prediction? The transplanted E29 cells:
a. Should go deep: P1 environment does not matter
b. Should go superficial: P1 environment does not matter
c. Should go deep, but in-culture conditions might affect fate
d. Should go superficial, but in-culture conditions might affect fate

The data:
superficial
deep
What do you conclude?
a. Signaling is required for blockage of deep layer fate
b. Signaling is required for blockage of superficial layer fate
c. Signaling is required to induce superficial layer fate

Possible Mechanism?
Different levels of
β-catenin activation at different stages
Signaling between the cells affects amount of b-catenin activation
Early stage: high b-catenin activation; later stage: no b-catenin activ

MCDB 4650 Class 11
Examples of cell fate commitment:
Eye development and Neural Crest Cells
Next exam is coming up on Monday Feb 25
Review session this Friday at 2:30 PM

What about other regions of the nervous system?

Sensory placodes are formed early
and are initially not committed
9.31
Eye itself is an outpocketing
of the Diencephalon
Vertebrate eye development
Source of Shh
Optic vesicle
(eye induction)

The eye field, and molecules expressed
Shh
(from notochord)

Shh splits the forebrain into two fields

Cell types of
the vertebrate
retina
9.34
RPE

To determine if retinal progenitors were multipotent,
they were labeled with a retrovirus that could be
visualized
All cells shown in the section of retina are likely descended
from a single progenitor – lineage tracing experiment
Is there any potential caveat?
So, progenitor cells are multipotent: ie, a single dividing cell
can give rise to several different kinds of cell types

Retinal cells differentiate in a certain order, with
considerable overlap in what is produced at what time
Retinal ganglion cells
Amacrines
Rods
Bipolar cells
Cook 2003

What about the environment of the retinal precursors (ie, signaling
ligands)? How can we test if it is involved in commitment of fate?
a. Isolate cells in culture from different stages of retinal
development
b. Do heterochronic transplants
c. Combine cell types from different stages of development in
culture
d. Isolate secreted proteins present in retina at different stages of
development and test their effect on cell fate
e. I want to do two of the above experiments

To test whether the fates could be
influenced, Cepko and colleagues (1999):
1. Verified what progenitor cells from
different stages made in isolated culture,
and
2. mixed the earlier stage progenitor cells
(E16) with later stage cells (P0), then
categorized what cell types were made.
Normal sequence: cones and amacrines, then rods

Retinal progenitor cells from two different stages were isolated and
put in culture:
• E16 (early) - labeled
• P0 (late)
• E16:P0 (two stages of cells mixed together with 20 fold excess
P0 )
THEN, just the E16 cells were followed to see what they
differentiated into

(from Belliveau and Cepko, 1999)
amacrines
1:20 ratio
Cones (GREY)
Rods (WHITE)
Cones (GREY)
Rods (WHITE)
A. % labeled cells that differentiated into
amacrine cells after 5 days in culture (5DIV)
B. % labeled cells that differentiated into
cone photoreceptors (grey) or rod
photoreceptors (white) after 5 days in culture
C. Same as B, but after 15 days in culture

Are the differentiated cells in the P0 extracts
influencing the fates of the progenitors?
E16 +P0
cells with
lots
amacrines
E16+P0 E16 + P0
cells with cells
few
amacrines
What can you conclude from this series of experiments?
% heavily labeled cells that
differentiated into
amacrines or photoreceptors
amacrine
photoreceptors

One possible model: there are two different sets of
progenitor cells that are not committed, but “biased”.
The signals from differentiated or even post-mitotic
cells changes the progenitor cells over time
Progenitors
Differentiated cells

Looking at markers for photoreceptors and bipolar cells in the retina:
Photoreceptor-
specific marker
Bipolar-specific
marker
early
late
From this data, the control over which cell type is produced could be
a. Transcriptional
b. Translational
c. Either

The microRNA stops being expressed over time
This micro RNA
specifically inhibits
the bipolarspecific
transcripts
from being
translated
(bright red label)

Take home:
Both intrinsic changes (decreasing levels of a
microRNA) and extrinsic changes (molecules secreted
by already differentiated cells) impact the
commitment of retinal progenitor cells at a given time
point.

Neural crest cells: unique cells of ectodermal origin that
ultimately populate many different locations and tissues

Neural crest cells: unique cells of ectodermal origin that
ultimately populate many different locations and tissues
Determination of
these cells as neural
crest requires BMP
which in turn
activates molecules
required for neural
crest specification
as well as migration

Different regions of
endoderm determine
different kinds of cranial
neural crest derivatives
(signals from endoderm to
crest cells):
transplant pharyngeal
endoderm that normally lies
right under the jaw to a host:
extra jaw is induced from
neural crest cells near the
transplant

On the other hand, there
are also signals from the
neural crest to the
pharyngeal endoderm:
if you transplant duck
neural crest cells into a
quail, the quail makes a
duck’s beak

This led to another interesting set of experiments:
Why don’t birds have teeth? (or conversely, why
do we have teeth rather than a beak?)
Teeth form from neural crest cells and adjacent
epidermis. The epidermis “coats” the tooth or the
beak, while the main part of each structure is
derived from neural crest cells.

teeth form from head neural crest cells and adjacent
epidermis: In mouse, the epidermis “coats” the tooth, while
the main part of the tooth is derived from neural crest cells
birds, who have these tissue types, the combination does not
lead to teeth.
what is the most likely explanation for why birds have lost the
ability to make teeth? Think about how things could have
changed on the molecular level.
mouse neural crest cells in isolation: teeth are not formed
bird neural crest cells in isolation: teeth are not formed
mouse jaw epidermis combined with bird neural crest:
teeth are not formed.
bird jaw epidermis combined with mouse neural crest:
teeth are formed.

The bird epidermis could be producing a signaling
molecule that can induce the neural crest cells to make
teeth, but the neural crest cells in the bird no longer
produce a functional receptor (or transcription factor
downstream) that allows them to respond. This could be
due to a mutation in the coding region or multiple
regulatory regions of the DNA that codes for the receptor
or transcription factor. Thus, when you put the bird
epidermis with mouse neural crest, since the receptor is
still made, teeth can form. The opposite could also be
true: that a signaling molecule released from the neural
crest is no longer present to induce the epidermis.

Summary: Neural crest potency
• Crest cells start out multipotent
• Some restriction exists initially (trunk cannot
make cardiac and cranial fates)
• Potency is restricted over time
• Fate largely determined by molecules in the
environment (primarily growth factors)
• Crest cells also influence the differentiation of
surrounding tissues (reciprocal induction)