Lecture 13

Question 1

What are the main roles of skeletal muscle

Answer 1

Control of coordinated movement and posture, communication through speech expression and writing, maintenance of temperature by heat release during contraction, and respiration

Question 2

Recall the pathway of muscle development

Answer 2

Stem cells –> myoblasts –> myotubes –> myofibres

Question 3

Describe the mature muscle cell

Answer 3

The mature muscle cell or myofibre is a multinucleate contractile cell

Question 4

Muscle cells aren’t differentiated until they become myofibres, T or F

Answer 4

F – the differentiated muscle cell is called a myotube, the myofibre is a mature muscle cell

Question 5

Myotubes are mononucleate, T or F

Answer 5

F – they are multinucleate

Question 6

How do myotube form

Answer 6

Fusion of mononucleate myoblasts

Question 7

At what stage in muscle development do the cells become committed to the muscle cell lineage

Answer 7

When they become myoblasts

Question 8

What are C3H10T1/2 cells

Answer 8

These are a population of fibroblast cells that are already capable of giving rise to myocytes under certain conditions

Question 9

What is the result of exposure of certain types of fibroblasts to 5aza and how does this occur

Answer 9

Exposure of the C3H10T1/2 cells to 5aza causes the fibroblasts to commit to a muscle cell fate. This is because 5Aza is a demethylating agent that demethylates histones and CpG nucleotides which results in a change in conformation of the chromatin. This provides a favourable environment for transcription and gene expression

Question 10

What does MyoD stand for

Answer 10

Myogenic determinant factor

Question 11

Explain the experimentation that lead to the isolation of MyoD

Answer 11

The transcriptomes of fibroblasts cells that have been exposed to 5Aza were compared to a population of the same cells that hadn’t been exposed to it to identify the different expression profiles of the cells that conferred their commitment to the muscle cell lineage. These mRNA transcripts were used to create cDNA which allowed for the isolation of MyoD

Question 12

What is meant by MyoD being a master regulatory gene and how was this discovered

Answer 12

MyoD can induce a transition of a fully differentiated cell into a fully differentiated cell. This was carried out by placing the MyoD gene under a constitutively active promoter and introducing the construct into terminally differentiated cells. This results in the cells switching to a fully differentiated muscle cell fate

Question 13

What type of protein is MyoD and how does it interact with DNA

Answer 13

MyoD is a basic helix-loop-helix protein that acts as a transcription factor. Its basic amino acid domain is responsible for binding to the DNA whilst the helix is involved in dimerization with E12 and E47 proteins. MyoD binds to E-box regions contained in the promoter and enhancer regions upstream of muscle related genes

Question 14

Other than MyoD what 3 other genes in the same family act to confer muscle cell fate

Answer 14

Myf5, myogenin, MRF4

Question 15

What is meant by the term myotome

Answer 15

The myotome is the dorsal region of the somite that will give rise to the skeletal muscles of the ventral trunk

Question 16

Initially the somitic mesoderm is pluripotent, T or F

Answer 16

F – it is multipotent and can give rise to cartilage, bone and skeletal muscle

Question 17

What is meant by the sclerotome and how is this region formed in the somites

Answer 17

The sclerotome is the region in the ventral somite that will give rise to the axial skeleton. It is formed when cells in the ventral somite undergo an EMT and delaminate

Question 18

How is the myotome formed

Answer 18

The myotome forms when dorsal epithelial cells in the somites undergo an EMT and drop beneath the somite

Question 19

What structures do the myotome give rise to

Answer 19

Skeletal muscle of the trunk (and limbs)

Question 20

Myotome cells become sandwiched between cells that remain epithelial in the dorsal somite called the dermomyotome and the cells of the ventral sclerotome, T or F

Answer 20

T

Question 21

What are the two definitive regions of the myotome and which specific structures do these give rise to

Answer 21

Cells in the medial myotome give rise to the epaxial muscles of the back. Cells in the lateral myotome will give rise to the hypaxial muscles of the abdomen and limbs

Question 22

MRFs are factors expressed in the myoblasts during embryogenesis with important functions in the formation of skeletal muscle, what region is their expression restricted to

Answer 22

The myotome

Question 23

What happens when you knockout Myf5 in mice

Answer 23

Mice produced are still viable but do show delayed myotome formation until MyoD is expressed

Question 24

What is seen in MyoD knockouts

Answer 24

Mice are born viable with no obvious muscle defects at birth. There is a slight delay in limb muscle development and a deficit in muscle regeneration in adults

Question 25

What is meant by functional redundancy

Answer 25

One gene can restore the function of another gene if that gene is knocked out

Question 26

What can be done to determine if Myf5 and MyoD act in functional redundancy

Answer 26

Double knockout mice

Question 27

What is seen in the MyoD and Myf5 -/- mice

Answer 27

Complete absence of skeletal muscle and no presence of myoblast cells

Question 28

What can be determined about how Myf5 and MyoD interact and dictate muscle generation

Answer 28

Myf5 and MyoD act in functional redundancy and are required to generate myoblast cells

Question 29

What is seen in myogenin knockout mice

Answer 29

The offspring die shortly after birth from a diaphragm defect. They also have reduced density of myofibres. The mice are capable of producing myoblasts but not myotubes

Question 30

What can be inferred about myogenin from its knockout in mice

Answer 30

Myogenin is required downstream of MyoD and Myf5 to confer muscle differentiation from myoblasts to myotubes

Question 31

Which genes are expressed during each stage of muscle cell development

Answer 31

MyoD, Myf5 and MRF4 expressed during specification of stem cells to a muscle lineage. Myogenin expressed during differentation. MRF4 expressed again during maturation

Question 32

Which region of the myotome gives rise to the muscles of the back

Answer 32

Epaxial myotome

Question 33

Which region of the myotome gives rise the muscles of the abdominal wall

Answer 33

Hypaxial myotome

Question 34

Which cell signalling events are required for formation of the epaxial myotome

Answer 34

Sonic hedgehog signalling from the notochord and floor plate and wnt signalling from the dorsal neural tube

Question 35

Which cell signalling events are required for formation of the hypaxial myotome

Answer 35

Wnt signalling from the ectoderm and BMP4 from the lateral mesoderm

Question 36

Which region contains cells that will give rise to the muscles of the limbs

Answer 36

Lateral myotome

Question 37

MyoD and Myf5 expression if delayed in the cells that become the muscles of the limb, explain the events that lead up to this delay

Answer 37

Cells in the lateral myotome undergo and EMT to initiate their migration. It is only once they have reached the dorsal and ventral positions in the limb bud where they begin to differentiate

Question 38

Describe the splotch mouse phenotype

Answer 38

Splotch mice have a loss of function of the Pax3 transcription factor and have neural tube and brain defects. Importantly for muscle development however, they have muscles of the trunk that develop normally but have a complete lack of skeletal muscles in the limbs

Question 39

What can be inferred about the action of Pax3 from splotch mice data

Answer 39

Pax3 acts upstream of MyoD in the cells of the somite that are to migrate and give rise to muscles of the limb

Question 40

How does Pax3 act

Answer 40

Pax3 is a transcription factor that is expressing in somitic cells and drives the expression of the c-Met receptor. c-Met binds to its ligand hepatocyte growth factor (HGF) which is synthesised by the mesenchymal cells of the limb. This acts as a chemoattractive signal to direct these cells to migrate to the limb bud

Question 41

Depletion of Pax3 results in a failure of limb skeletal muscle cell progenitors to migrate to the limb bud, T or F

Answer 41

T

Question 42

What is the name given to muscle stem cells that are retained in adult muscles and dictate muscle regeneration

Answer 42

Satellite cells

Question 43

What is significant about the number of muscle stem cells during development and what role does this indicate

Answer 43

Satellite cells make up 32% of muscle nuclei at birth but this decreases to only 5% by adulthood. This indicates the role of these cells in directing postnatal muscle growth

Question 44

What stimuli can trigger the induction of muscle stem cells in adults

Answer 44

Injury, exercise, denervation and stretching. They can also become more active in muscle wasting disease such as Duchene muscular dystrophy

Question 45

What is significant about the gene expression by muscle specific stem cells in muscle regeneration

Answer 45

They express the same sequence of genes as is seen in development and embryogenesis firstly expressing Pax7(duplicate of Pax3) then Myf5, MyoD and Myogenin

Question 46

What signalling pathway is responsible for induces MyoD expression and causing muscle differentiation

Answer 46

Sonic hedgehog