Myogenesis

Question 1

List the functions of skeletal muscle

Answer 1

Movement and posture
Communication
Body Temperature maintenance
Respiration

Question 2

Give two examples of muscle wasting diseases

Answer 2

Duchenne dystrophy

Becker’s dystrophy

Question 3

What is Duchenne dystrophy?

Answer 3

Progressive muscle degeneration
Onset 3 to 5 year olds
Mainly affects boys as it is X-linked recessive

Question 4

What is Becker’s dystrophy?

Answer 4

Variant of duchenne dystrophy

Voluntary muscles function slightly better than in duchennes

Question 5

What are the steps in making a muscle?

Answer 5

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

Question 6

What usually dictates the speed of a muscles metabolism?

Answer 6

Their innervation

Question 7

Who isolated MyoD?

Answer 7

Weintraub 1987

Question 8

What is 5Aza?

Answer 8

A demethylating agent which causes histone demethylation

Question 9

Which cell line was identified to sometimes turn into myoblasts if treated with 5Aza?

Answer 9

Fibroblats C3H10T1/2

Question 10

If demethylatng agents are applied to a cell what is the effect?

Answer 10

The depress the state of the chromatin in the cell

Question 11

What did Weintraub do in his experiment?

Answer 11

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

Question 12

What is MyoD referred to as?

Answer 12

A master regulatory gene

Question 13

What happens if MyoD is put into any cell type?

Answer 13

The cell will be converted into a myoblast as it is a master regulator

Question 14

Which other proteins are in the MyoD family?

Answer 14

MyoD, Myf5, Myogenin, MRF4

Question 15

What do bHLH proteins stand for?

Answer 15

Basic helix loop helix

Question 16

What is a bHLH?

Answer 16

A protein structural motif that characterises one of the largest families of dimerising transcription factors

Question 17

Which cofactors dimerise with the helix - loop - helix domain?

Answer 17

E12 and E47

Question 18

What are the three functions of the MyoD protein families?

Answer 18

1) Transcription activator
2) Form heterodimers with E12 or E47
3) Binds to E boc: CANNTG

Question 19

Where does skeletal muscle originate from?

Answer 19

The dermyotome

Question 20

Skeletal muscle progenitors express which paired box transcription factor?

Answer 20

PAX3

Question 21

In the trunk PAX3 positive cells contribute to what?

Answer 21

The myotome

Question 22

What are the two myotome domains?

Answer 22

Epaxial (medial)

Hypaxial (lateral)

Question 23

When are MRFs expressed?

Answer 23

In myoblasts during embryogenesis

Question 24

In an embryo stained for MRF what does the staining correlate to?

Answer 24

To where the skeletal muscle is forming

Question 25

Which is the earliest gene to be expressed from the MyoD protein family and when is it expressed?

Answer 25

Myf5 | 8 days of gestation (somites form at 7.5 days)

Question 26

How are genes targeted in ES cells?

Answer 26

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

Question 27

What happens in a Myf5 knockout?

Answer 27

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

Question 28

What happens in a MyoD knockout?

Answer 28

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

Question 29

What happens when both MyoD and Myf5 are knocked out?

Answer 29

Complete absence of skeletal muscles, no presence of myoblasts Shows the you need one of them to generate myoblasts

Question 30

What happens in a myogenin knockout?

Answer 30

Mice die shortly after birth from diaphragm defect Rediced density of myofibers replaced by myoblasts Shows myogenin is required for muscle differentiation

Question 31

Which genes are involved for a Pax3 positive cell to be determined to form a myoblast?

Answer 31

Myf5 MyoD MRF4

Question 32

Which gene is required for a myoblast to differentiate into a myotube?

Answer 32

Myogenin

Question 33

Wghcih gene is required for a myotube to mature into a myofiber?

Answer 33

MRF4

Question 34

Which two signalling pathways are involved in controlling muscle gene activation in the epaxial myotome?

Answer 34

Wnts - come from the notochord | Shh - comes from the dorsal part of the neural tube

Question 35

Which two signalling pathways are involved in controlling muscle gene activation in the hypaxial myotome?

Answer 35

Wnts | BMPs - lateral plate mesoderm

Question 36

Why does a PAX3 mutant mouse have a white spot on their belly?

Answer 36

PAX3 is important for neural crest formation and therefore pigmentation

Question 37

PAX3 mutant mice show what phenotype in the limbs?

Answer 37

They lack muscles in the limbs

Question 38

How do muscle cells migrate into the limb bud?

Answer 38

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

Question 39

PAX7 knockouts do not have what ability?

Answer 39

To repair muscles in the adult

Question 40

When are satellite cells visible in mice?

Answer 40

E17.5 in the limbs

Question 41

Where are satellite cells seen?

Answer 41

Located under the basal lamina (inbetween the basal lamina and the muscle fibres

Question 42

What happens to our satellite cells over time?

Answer 42

The amount we have decreases due to the postnatal growth of muscles

Question 43

What activates satellite cells?

Answer 43

Injury causes themm to be activated due to reactive oxygen species

Question 44

Satellite cells represent the what percentage of muscle cells in the embryo and the adult

Answer 44

32% embryo | 5% adult