Download Determination and Differentiation

BIOLOGY 205/SECTION 7
DEVELOPMENT- LILJEGREN
Lecture 7
Determination and Differentiation
1. DETERMINATION vs. DIFFERENTIATION vs. FATE vs. SPECIFICATION
a. We’ve already talked about cells having a certain FATE, that is often specified or
modulated by induction events. A level of commitment beyond cell fate is
specification. A specified cell has been told what it should be. If it is
experimentally isolated, it will follow those instructions; however, its fate can still
be influenced by different environments. Beyond cell specification is
determination. At this level of commitment, the cell cannot be made to change
fates by a different environment, that cell is said to be DETERMINED. A cell is
said to be determined if it has made an irreversible (usually)
developmental choice among options. A more rigorous definition is that a cell
is determined when it has undergone an internal and self-perpetuating change
distinguishing itself & its descendents from other cells and committing to a
specialized course. This concept differs from DIFFERENTIATION because that
refers to the elaboration of the fate of a cell. Differentiation is overt cell
specialization: a cell gaining new detectable differences. In other words, a
cells knows what it will become (it is determined) before it actually becomes that
type of cell (it differentiates).
b. Another aspect of determination is that the fate decision becomes selfperpetuating, so that the subsequent progeny of the determined cell usually
have the fate (sometimes in modified form) of that cell.
2. Evidence for determination during development.
a. Transplant experiments (Spemann 1918): showed changes in cell determination
1. Experiment #1: If take an amphibian embryo at early gastrulation and
transplant prospective epidermis (skin) where neural tube would normally
form, the transplant tissue forms neural tube and brain. If you reverse the
experiment, and put prospective neural plate where skin usually forms, it will
form skin. THEREFORE AT THIS STAGE OF DEVELOPMENT BOTH FATES CAN
BE SWITCHED BY CHANGING THE ENVIRONMENT. THE CELLS ARE NOT YET
DETERMINED.
2. Experiment #2: The same experiment done during a later stage of
gastrulation, gives a very different result. At this point prospective epidermis
will make epidermis even if it is put where neural plate normally develops.
Likewise the prospective neural plate will form neural plate at a epidermal
location. THEREFORE AT THIS STAGE BOTH FATES CANNOT BE SWITCHED
BY CHANGING ENVIRONMENT, AND THE CELLS ARE DETERMINED.
However, when the experiment was done, the cells had not yet differentiated
into epidermis or neural plate.
b. The ultimate transplantation experiment: CLONING.
•
Somatic nuclear transfer: replace nucleus of unfertilized egg with
that of differentiated somatic cell.
•
Most famous cloned animal is Dolly, but the cloning process was
actually developed in frogs back in the 1950s
•
How does cloning work?
•
While occasionally this works at a low frequency so that an entire
organism is regenerated, usually this experiment does not succeed!
Furthermore, as development proceeds the ability of cells to alter fate
progressively decreases. Remember that many clones are abnormal, and/or
develop lots of problems.
3. Determination= heritable changes in gene expression. How does it occur?
a. Ultimately changes must be heritable (on a cellular level), and must involve
changes in gene expression because this regulates machinery available to a cell to
carry out specialized tasks.
b. With few exceptions, determination occurs as a result of selective gene
EXPRESSION without changes in the DNA content or gene arrangement. Selective
gene expression appears to result from a combination of (1) information in the
cytoplasm of the cell that gets asmmetrically allocated to daughter cells, (2) basechange modifications of the DNA that influence expression of genes (ie. DNA
methylation), (3) non base-change modification of the DNA (ie. post-translational
modification of histone proteins) .
c. The most impressive evidence for cytoplasmic contributions comes from cloning,
which demonstrates that the cytoplasm of the zygote can reprogram the
nucleus of the differentiated cell back to the starting point.
4. Global mechanisms of regulating gene expression. Heritable changes in the state of
the DNA results in determination.
a. Methylation. Highly methylated DNA is found in mammals (but NOT in many
other animals). Certain enzymes can methylate cytosine, and the methylated DNA
is usually not amenable to transcription into RNA. It is thought that the
methylated DNA is packaged into larger, tighter chromatin structures that make
the DNA inaccessible to RNA polymerase. Methylation is heritable--maintenance
methylases methylate "daughter" DNA molecule in the same pattern as "parent"
DNA molecule.
b. “DNA Imprinting” of egg and sperm DNA is an example of how changes in DNA
methylation lead to changes in gene expression. Causes an egg to develop with
only the maternal or paternal copy of a gene being active, not both copies.
Mechanisms—one parental copy becomes permanently inactivated by DNA
methylation. More than 70 genes in mammals known to be imprinted!
c. Examples of DNA imprinting that affect fetal growth.
(1.) Insulin growth factor 2 (Igf2). Normally this protein stimulates rapid growth
and is specifically expressed during fetal development. Only the paternal copy
of the corresponding gene is transcribed.
(2.) Insulin growth factor 2 receptor (Igf2r). This protein inhibits Igf2 activity and
represses fetal growth. Only the maternal copy of the corresponding gene is
transcribed.
(3.) If Igf2 from dad knocked out (or both copies mutant), mice are born 40%
smaller than normal
(4.) If Igf2 copy from mom NOT imprinted, get fetal overgrowth. In humans, this
is the cause of Beckwith-Weidermann syndrome.
(5.) If Igf2r from mom knocked out, fetus is 30% larger than normal and dies late
in gestation
d. Chromatin structure. DNA in the nucleus is covered with proteins (“string
on the beads”.
1. Chromatin=complex of DNA and histone proteins
2. Depending on their packing arrangement, these proteins repress expression of
unwanted genes or maintain expression of needed genes.
3. Histone proteins have mechanisms to resume binding after DNA replication.
4. Post-translational modification of histone proteins (methylation, acetylation or
phosphorylation) plays a critical role in regulating gene expression. Ie. lack of
acetylation contributes to X-inactivation of one X chromosome. In figure, see
chromosomes from a fibroblast cell stained with fluorescent antibody to
acetylated histone H4, and only one chromosome (X) is not stained, indicating
that it has low levels of acetylation. Correlation of acetylation with gene
expression.
5. myoD: A master regulatory protein—or is it?
a. Experiment#1- Reversing determination
Take tissue culture cell line that has properties of fibroblast cells (connective tissue).
Grow in azacytidine- this leads to demethylation of DNA
Individual cells switch fates; they & descendents can form fat cells, muscle cells, bone
cells etc.
b. Experiment#2- What genes are demethylated to cause a switch in cell fate?
Remember if treat cells with azacytidine it can lead to demethylation of DNA. So in
this experiment, fat cells were treated with azacytidine, so they no longer have a fat
cell fate. Instead a fraction of them can now go the muscle cell fate route. Myoblasts
are committed muscle cell precursors, they are determined but not yet differentiated.
Chromatin (DNA packaged in proteins) from these myoblast cells was isolated and cut
into pieces, then the different pieces were introduced (transfected) into different
untreated fat cells: a fraction of these cells were able to switch fate and become
muscle cells. This indicates that those cells that were able to switch fate got a unique
piece of chromatin that contained a gene essential for specifying muscle cell fate,
which turned out to be myoD.
ie. myoblasts contain active genes that control muscle cell fate.
Ultimately a single gene was isolated that conferred this property= myoD
Resulting theory- myoD is the master regulator of muscle development. If
express myoD under the control of a viral promoter and introduce into fat cells, it can
convert them to muscle cells. However, just because a molecule can do something in
vitro doesn't mean it does the same thing in vivo (in the embryo)!
c. Experiment #3- Is myoD expressed at appropriate time to regulate muscle?
Use radioactive probes to examine when myoD is expressed in frog embryo.
Result- yes, myoD is expressed during muscle differentiation. i.e. It's in right place
at right time!
d. Experiment #4- What does the myoD protein do?
myoD bHLH (basic helix loop helix) protein resembles other "transcription factors"
which turn genes on or off.
Does myoD bind to the DNA of the right sort of genes?
Measure ability of myoD protein to bind to muscle specific genes
Result- most muscle specific genes examined have binding sites for myoD
e. Experiment #5—Is myoD is the master regulator of muscle development?
Eliminate myoD gene from mice- Do they make muscle?
Result- oops! They are normal- Make new theory!
New theory- myoD and related proteins- myf5, myogenin, etc.
cooperate to regulate muscle development.
f. Experiment #6—Are myoD and myf5 required for muscle development?
Make mice lacking both myoD and myf5 bHLH transcription factors.
Result- YES! animals now lack skeletal muscle! Proof of redundancy
6. Cellular memory and the engrailed gene in Drosophila.
a. A cell fate choice in Drosophila- posterior vs. anterior in the segment
b.
c.
d.
e.
1) Remember, anterior cells make hairs & posterior cells do not, and wingless is one
of the signaling molecules that tells cells to be posterior.
2) How is this choice regulated and remembered for rest of life?
Early in Drosophila development, a set of transcription factors turn on the engrailed
gene specifically in posterior cells. Then these transcription factors are turned off.
How does engrailed remain on in some places and off in others?
Mechanism #1- cell:cell signaling.
1) Neighboring cells produce and secrete Wingless signal.
2) Engrailed-expressing cells must receive Wingless signal in order to continue to
express Engrailed.
3) How do we know this? In embryos lacking Wingless, engrailed is turned on
correctly but then turns off.
Mechanism #2- autoregulation
1) engrailed encodes transcription factor that binds to DNA near its own gene &
keeps it on where it is already on=autoregulation.
2) How do we know this? In mutant embryos with only non-functional Engrailed
protein engrailed gene expression comes on correctly and then goes off.
Mechanism #3- A specialized set of chromatin-binding proteins
1) engrailed remains on in some cells, off in other for rest of life cycle ≥60 days.
2) If autoregulation explains how it stays on, what explains how it stays off?
3) Theory- several hours after gastrulation a special set of chromatin-binding
proteins (Polycomb proteins) bind to the histone-wrapped engrailed gene in
cells where it is turned off. These proteins are thought to help wrap up the DNA in
a way that keeps it off forever.
4) How do we know this? Scientists removed gene for one of the Polycomb proteins
in the adult fruit fly, which caused the engrailed gene to come back on in cells
where it had never been on.
5) In contrast, other chromatin-binding proteins (Trithorax proteins) help keep
genes on by modifying histone-wrapped genes (nucleosomes) to keep them
accessible to transcription factors and RNA polymerase.