Myogenesis Flashcards
List the functions of skeletal muscle
Movement and posture
Communication
Body Temperature maintenance
Respiration
Give two examples of muscle wasting diseases
Duchenne dystrophy
Becker’s dystrophy
What is Duchenne dystrophy?
Progressive muscle degeneration
Onset 3 to 5 year olds
Mainly affects boys as it is X-linked recessive
What is Becker’s dystrophy?
Variant of duchenne dystrophy
Voluntary muscles function slightly better than in duchennes
What are the steps in making a muscle?
1) Stem cells have to be specified to differentiate
2) Muscle progenitor cells differentiate = MYOBLAST
3) Differentiated muscles cells express enzymes and contractile elements to form myotubes
4) Maturation of myotubes to myofibers
What usually dictates the speed of a muscles metabolism?
Their innervation
Who isolated MyoD?
Weintraub 1987
What is 5Aza?
A demethylating agent which causes histone demethylation
Which cell line was identified to sometimes turn into myoblasts if treated with 5Aza?
Fibroblats C3H10T1/2
If demethylatng agents are applied to a cell what is the effect?
The depress the state of the chromatin in the cell
What did Weintraub do in his experiment?
1) He took 5 Aza treated and untreated fibroblasts and converted their mRNA to cDNA
2) He then hybridised the two cDNA populations together to identify the muscle specific genes from the house keeping genes
3) Where there are two copies of genes from each population the DNA hybridises, but where there isn’t it remains single stranded
4) This is how he identified MyoD cDNA
What is MyoD referred to as?
A master regulatory gene
What happens if MyoD is put into any cell type?
The cell will be converted into a myoblast as it is a master regulator
Which other proteins are in the MyoD family?
MyoD, Myf5, Myogenin, MRF4
What do bHLH proteins stand for?
Basic helix loop helix
What is a bHLH?
A protein structural motif that characterises one of the largest families of dimerising transcription factors
Which cofactors dimerise with the helix - loop - helix domain?
E12 and E47
What are the three functions of the MyoD protein families?
1) Transcription activator
2) Form heterodimers with E12 or E47
3) Binds to E boc: CANNTG
Where does skeletal muscle originate from?
The dermyotome
Skeletal muscle progenitors express which paired box transcription factor?
PAX3
In the trunk PAX3 positive cells contribute to what?
The myotome
What are the two myotome domains?
Epaxial (medial)
Hypaxial (lateral)
When are MRFs expressed?
In myoblasts during embryogenesis
In an embryo stained for MRF what does the staining correlate to?
To where the skeletal muscle is forming
Which is the earliest gene to be expressed from the MyoD protein family and when is it expressed?
Myf5
8 days of gestation (somites form at 7.5 days)
How are genes targeted in ES cells?
1) electroporate the cell with mutated gene
2) Select cells which have taken up the DNA
3) Analyse colonies using gel electrophoresis
4) Make chimeras
5) Implant into the the mother
6) Test offspring for chimerism
7) Test germline for transmission
8) Cross the heterozygotes to analyse the offspring for the phenotype
What happens in a Myf5 knockout?
Mice are viable
No obvious muscle defect at birth
During embryogenesis there is a delay in the myotome formation until the expression of MyoD
Myf5-/- cells migrate aberrantly into sclerotome and dermatome
What happens in a MyoD knockout?
Mice are viable
No muscle defect at birth
During embryogenesis increases Myf5 expression in somites to compensate
Slight delay in limb muscle development
Deficit in muscle regeneration in adult mice
What happens when both MyoD and Myf5 are knocked out?
Complete absence of skeletal muscles, no presence of myoblasts
Shows the you need one of them to generate myoblasts
What happens in a myogenin knockout?
Mice die shortly after birth from diaphragm defect
Rediced density of myofibers replaced by myoblasts
Shows myogenin is required for muscle differentiation
Which genes are involved for a Pax3 positive cell to be determined to form a myoblast?
Myf5
MyoD
MRF4
Which gene is required for a myoblast to differentiate into a myotube?
Myogenin
Wghcih gene is required for a myotube to mature into a myofiber?
MRF4
Which two signalling pathways are involved in controlling muscle gene activation in the epaxial myotome?
Wnts - come from the notochord
Shh - comes from the dorsal part of the neural tube
Which two signalling pathways are involved in controlling muscle gene activation in the hypaxial myotome?
Wnts
BMPs - lateral plate mesoderm
Why does a PAX3 mutant mouse have a white spot on their belly?
PAX3 is important for neural crest formation and therefore pigmentation
PAX3 mutant mice show what phenotype in the limbs?
They lack muscles in the limbs
How do muscle cells migrate into the limb bud?
Mesenchymal cells from the limb bud secrete HGF/SF
PAX3 induces expression of c-met, the receptor for HGF/SF, in the limb muscle progenitor cells
Following c-met induction, cells migrate into the limb
PAX7 knockouts do not have what ability?
To repair muscles in the adult
When are satellite cells visible in mice?
E17.5 in the limbs
Where are satellite cells seen?
Located under the basal lamina (inbetween the basal lamina and the muscle fibres
What happens to our satellite cells over time?
The amount we have decreases due to the postnatal growth of muscles
What activates satellite cells?
Injury causes themm to be activated due to reactive oxygen species
Satellite cells represent the what percentage of muscle cells in the embryo and the adult
32% embryo
5% adult
