Transcript Determination and Differentiation

Induction: information from
neighbors influences cell fate
responder
inducer
If induction tells cells
what to do,
how do they remember
these instructions
for a lifetime??
DETERMINATION
FATE: what a cell will become at a later time--its future!
DIFFERENTIATION: the actual elaboration of a cell’s fate
DETERMINATION: internal and self-perpetuating changes that
distinguish a cell & its descendents from other cells and commit
them to a specialized course
In other words, a cell is set to become a certain kind of cell
before it actually becomes that type of cell.
And usually there’s no turning back.
This concept differs from fate in that for a time a cell’s fate can
often be changed by changing environment; it differs from
DIFFERENTIATION because this is overt cell specialization.
Determination can be assayed by
transplantation experiments
Early
gastrula
Cells alter their fate
to match that of
neighbors
i.e. not determined
Transplant
nervous system
to epidermis
and vice versa
Late
gastrula
Cells retain their
original fates,
regardless of
neighbors
i.e. determined
(see also Figure 10.18)
Cloning:
the ultimate
transplant
experiment
Figure 4.8
Dolly (and Bonnie)
Cloning:
The ultimate transplant experiment
Freddy, a cloned leopard frog (Rana pipiens)
Figure 4.5
How does
cloning work?
Cloning success drops dramatically as
the age of the nuclear donor increases
Figure
4.6
DETERMINATION
results from heritable
changes in gene expression
All cells have the same genes yet
different cells make different mRNAs
Non-base change modifications of DNA result
in selective gene expression
Evidence for
cytoplasmic
contributions
to selective gene
expression
DNA Methylation is used in mammals to
keep unneeded genes inactive
Bone-specific
genes are
methylated
CH3 CH3 CH3
CH3
CH3
Skin-specific
genes are not
methylated
Skin cells
Muscle-specific
genes are
methylated
Gene
expression
CH3 CH3 CH3
CH3
Methylation of DNA
Figure 5.19
DNA Methylation is used in mammals to
keep unneeded genes inactive
Bone-specific
genes are
methylated
CH3 CH3 CH3
CH3
CH3
Skin-specific
genes are not
methylated
Skin cells
Muscle-specific
genes are
methylated
Gene
expression
CH3 CH3 CH3
CH3
Imprinting results from pre-programmed
differences in DNA methylation of
selected genes in the sperm and egg
Genomic imprinting occurs in most mammals
*
*
*
Igf2r
“A Silent Struggle”, New York Times, March 14, 2006
Igf2
Imprinted genes
affect fetal growth
Insulin growth factor 2
(Igf2)
growth
Insulin growth factor 2
receptor (Igf2r)
growth
What happens if dad’s
copy of Igf2 is deleted?
Insulin growth factor 2
(Igf2)
growth
Insulin growth factor 2
receptor (Igf2r)
growth
What happens if mom’s
copy of Igf2 isn’t imprinted?
Beckwith-Weidemann syndrome
Insulin growth factor 2
(Igf2)
Insulin growth factor 2
receptor (Igf2r)
overgrowth
growth
What if mom’s copy
of Igf2r is deleted?
Insulin growth factor 2
(Igf2)
growth
Insulin growth factor 2
receptor (Igf2r)
growth
What happens if mom’s
copy of Igf2 is deleted?
Insulin growth factor 2
(Igf2)
growth
Insulin growth factor 2
receptor (Igf2r)
growth
DNA within cells is not naked!
DNA is wrapped around histone proteins as chromatin
Figure 5.1
Inactive genes are thought to be wrapped up
in an inactive "chromatin conformation"
Bone-specific
genes are
wrapped up and
inaccessible
Skin cells
Skin-specific
genes are not
wrapped up and
so are accessible
Muscle-specific
genes are
wrapped up and
inaccessible
mRNA
Gene
expression
X chromosome inactivation:
Silencing one of the two X’s in female cells
Xist RNA is associated
with the silenced
X chromosome
Post-translational
modification of
histones plays
a key role
in regulating
gene expression
X chromosome
inactivation
(See also Figure 5.25)
myoD
Experiment #1
By altering methylation,
cells can be reprogrammed
Bone cells
Muscle cells
"Fibroblast-like"
mouse cells
Fat cells
 Treat with 5-azacytidine to reduce DNA methylation
Experiment #2
Chromatin transferred from myoblasts
can transform untreated fat cells.
Transformation frequency suggests
only one or few of the genes have this ability
Ultimately a single gene was identifiedmyoD
-whose expression could convert
any other cell type into muscle cells
MyoD is expressed at the right place
and time to be a master regulator of
muscle development
Somites
Experiment #3
Experiment #4
Does the myoD transcription factor bind DNA sites
within the promoters of the expected gene types?
Bone-specific genes lack
myoD binding sites
Muscle-specific genes have
myoD binding sites
Fat-specific genes lack
myoD binding sites
OFF
myoD
ON
muscle-specific
mRNA
myoD site
OFF
Experiment #5
Scientists "knocked out" the myoD gene to test its
predicted role in muscle development
Eliminate gene encoding myoD
Does mouse develop muscles?
!!
Experiment #6
MyoD and related transcription factors
work together to regulate muscle cell fate
How do cells remember their decisions?
Cellular memory and the engrailed gene
Remember that
Wingless is
expressed by
specific cells
within each segment
Engrailed is a
transcription factor
expressed by cells
posterior to those that
express Wingless.
Anterior
Posterior
It defines the posterior
boundary of each
segment.
Wg En
Here’s the real thing: engrailed
Mechanism #1:
cell-cell signaling
Engrailed expression
requires Wingless
signaling by the
neighboring cells.
Mechanism #2: autoregulation
Later on, engrailed becomes autoregulatory
ie. it turns on its own synthesis
Segment
boundary
en
ON
en gene
Segment
boundary
More engrailed
protein
Mechanism #3: specialized chromatin proteins
In cells where engrailed is turned off, a set of specific chromatin
proteins bind to it and wrap it up in a permanently inactive state.
Segment
boundary
Segment
boundary
OFF
en gene
Polycomb group
proteins turn off
engrailed in cells
it’s not needed.
en gene
Permanently
OFF