lecture 13 - myogenic lineage Flashcards
What experiment supports the idea of regulative development?
Hans Driesch (1892) : cells in the early (sea urchin) embryo can be separated
from each other and individual cells give rise to fully developed sea urchins. This
means that each cell in the early embryo must contain all the necessary information
for normal development: totipotent.
What is Pre-implantation genetic diagnosis?
following in vitro fertilization a single cell is
removed from an embryo and is tested for
gene mutations; if there is no evidence of a
defect, the embryo is implanted to give rise
to a healthy baby. Regulative development ,
and the totipotency of the cells, allows the
rest of the embryo to compensate for the
missing cell.
What experiment shows gene constancy?
Somatic cell nuclear transfer (SCNT) ‘cloning’
John Gurdon (1962), Ian Wilmut (1996)
A fully differentiated adult cell was used to generate the
first cloned mammal. This shows that nothing in the
genome is lost when cells become specialised/differentiated.
“Gene constancy”
Describe differentation
The fertilised egg is totipotent.
Early embryonic cells appear identical and are totipotent/pluripotent.
During development, cells become restricted in what they can become.
In the end, over 200 different cell types differentiate in a mammal.
Differential gene expression is the basis for the emergence of differentiated cell types.
Cell signalling changes patterns of gene expression that are required for the emergence of differentiated cell types.
Cell lineage determination and differentiation are best understood in the skeletal muscle cell lineage.
What are somites?
In vertebrates, all skeletal muscle in the body is
derived from the somites.
Somites are segmented blocks of paraxial mesoderm.
New somites form from the anterior end of the pre-segmental plate mesoderm at regular intervals.
What is somitogenesis?
When the somite is first formed, the cells are unspecified. Later, regions of the somites become committed to forming only certain cell types. The parts of the somites are:
The scleratome: forms in the ventral medial part of the somite. The cells of the sclerome undergo mitosis, lose their epithelial structure and become mesenchymal cells. This part of the somite will differentiate as chondrocytes and give rise to the ribs and vertebrae.
The dermamyotome forms in the dorsal part of the somite and goes on to form:
the dermatome which is the dorsal most layer of the somite and will give rise to the mesenchymal layer of the dorsal skin (the dermis)
the myotome which will give rise to the deep muscles of the back and other muscle cells migrate from the ventral lateral part of the myotome to form the muscles of the bodywall and the limb.
All skeletal muscle in the vertebrate body is derived from the somites.
How is myogenesis activated?
The skeletal muscle cell lineage is specified in a subset of somite cells.
Signalling molecules are secreted by nearby structures such as the neural tube, notochord, dorsal ectoderm and lateral plate mesoderm.
These signals act together to activate myogenesis in the dorsal medial somite cells.
The signals do this by activating the expression of regulatory genes that direct cells down the skeletal muscle lineage.
What is myogenesis?
skeletal muscle development
Myoblasts are undifferentiated, proliferative cells within the somite committed to forming skeletal muscle.
Myoblasts can be isolated from embryos and kept in culture where they proliferate; but will differentiate as muscle when growth factors are removed .
Growth factors in the media allow myoblasts to proliferate.
Removal of growth factors allows myoblasts to differentiate.
When myoblasts differentiate they: (i) stop dividing (ii) fuse to form multinucleated myofibre and (iii) express contractile protein genes
Observation: when myoblasts stop dividing and fuse, they activate the expression of
a very large number of different genes all at the same time -> Points to a common transcriptional regulator.
Describe how experiment points to a dominant regulator of myogenesis
When a human liver cell is fused with a mouse muscle cell, the expression of human, muscle specific genes is detected.
This means the human nucleus, that was expressing liver genes, is now expressing muscle genes.
This suggests that there is something in the muscle cell that can activate transcription of muscle genes.
Points to a dominant regulator of myogenesis.
A mouse fibroblast cell line was treated with a drug that hypomethylates DNA.
50% of these cells converted to myoblasts.
Hypomethylation removes methyl groups from DNA; these methyl groups are usually found in regions of DNA that are not transcribed and removing them activates gene expression.
The number of fibroblasts that convert to myoblasts (50%) is consistent with the activation of a single locus. Points to a dominant regulator of myogenesis.
A comparison of the mRNAs present in the fibroblasts to the mRNAs present in the 5-aza-myoblasts, identified a cDNA capable of converting fibroblasts to myoblasts: it was named MyoD.
What is MyoD?
MyoD was the first identified member of a family of bHLH transcription factors that regulate the expression of skeletal muscle genes. The other members are myf5, myogenin and MRF4. They bind specific DNA sequences (called E-boxes) to activate gene expression.
The bHLH transcription factors coded for by the myogenic regulatory genes are collectively referred to as the MRFs (myogenic regulatory factors).
These regulators are expressed very early and specifically in the skeletal muscle cell lineage in all vertebrates.
MyoD and myf5 are expressed very early in somite cells.
Myogenin and Mrf4 are expressed later.
The MRF genes are expressed exclusively in cells in the myogenic lineage.
Describe the MRF regulated pathway
Signals from surrounding tissues activate the expression of MRFs in the early somite
MyoD and Myf5 are muscle determination genes.
Proliferating myoblasts express MyoD and Myf5 prior to differentiation
Myogenin regulates myoblast fusion and muscle differentiation
How is myogenesis regulated?
MRFs are basic helix-loop-helix transcription factors.
MRFs heterodimerize with other bHLH transcription factors called E-proteins (such as E12)
This dimer binds to an E-box , a regulatory sequence found in the enhancers of targets genes.
An E-box consists on a sequence of “CANNTG”
The genes transcriptionally activated by MRFs include contractile protein genes like actin, myosin, troponin…etc…as well as MRFs.
How do MRFs regulate skeletal muscle development?
MRFs are able to activate the whole myogenic programme (ie, transform non-muscle cells into muscle cells).
MRFs are bHLH transcription factors
MRFs activate the expression of skeletal muscle specific genes by binding to E-boxes present in their enhancer/ promoter s
MRFs auto- and cross-regulate MRF expression (by binding E-boxes)
MRFs are expressed early and specifically in cells of the myogenic lineage (ie, somites, myotome)
MRFs are key regulators of muscle development in all vertebrates
A mouse knock-out, lacking myoD and myf5 has no skeletal muscle (ie they are essential for muscle development).
