Lecture 12: myogenesis

Question 1

Describe the general pathway of muscle formation.

Answer 1

Stem cells → specification → myoblast (muscle progenitor) → differentiation → myotube → maturation → myofibril

Question 2

How was MyoD identified?

Answer 2

MyoD is a myogenic determination gene.

When fibroblast stem cells were cultured in the presence of 5AZA, a demethylating agent, they became myoblasts. 5AZA is a demethylating agent through which DNA releases heterochromatin and genes are activated that convert them to myoblasts. To understand this:

1. treat one set of fibroblast cells with 5AZA and leave another as WT
2. isolate the mRNA from both → convert to cDNA through reverse transcriptase
3. subtract the two cDNA populations to obtain the section of DNA with muscle specific genes
4. screen the cDNA using probes generated from myoblasts

The gene identified through this method was MyoD

When placed downstream of a powerful promotor sequence in non-muscle cells they eventually become a myotube with muscle-specific proteins.

Question 3

Discuss the family of proteins that MyoD belongs to.

Answer 3

bHLH proteins (basic Helix-Loop-Helix proteins)

These proteins:

• have a basic DNA binding domain
• have a HLH dimerisation domain - dimerises with E12 and E47 proteins
• are transcriptional activators that bind to the Ebox of a muscle gene
• also include MyoD, Myf5, Mrf4 and myogenin
• are activated by Pax3

The Ebox contains a CANNTG sequence i.e., C, A, Any nuclotide, Any nucleotide, T, G.

Question 4

Explain the results of MyoD, Myf5 and myogenin knockout studies in mice. What are the conclusions from these?

Answer 4

Myf5 KO = Mice viable, no obvious defects at birth. Delay in myotome formation until onset of MyoD expression. The mutant cells migrate into sclerotome and dermatome.

MyoD KO = Mice viable, no obvious defects at birth. Slight delay in limb muscle development and deficit in muscle degeneration in adult mice.

Myf5/MyoD KO = complete absence of skeletal muscles, no presence of myoblasts.

Conclusion: Myf5 OR MyoD is required to generate myoblasts. REDUNDANCY.

Myogenin KO: = Mice die shortly after birth due to a diaphragm defect i.e., can’t breathe. Reduced density of myofibres. More myoblasts.

Conclusion: Myogenin is required for muscle differentiation. Therefore referred to as a muscle differentiating factor and is only found in non-dividing cells.

Question 5

Where do skeletal muscles arise?

Answer 5

The paraxial mesoderm forms a pair of somites next to the neural tube. The dorsal somite remains epithelial and forms the dermomyotome. The dermomyotome contains all skeletal muscle progenitor cells which express TF PAX3.

There are two domains of myotome:

1. Medial → muscles of the back
2. Hypaxial → muscles of the body wall and limb

Question 6

Explain how the medial myotome is specified, differentiated and patterned in the embryo. Draw the pathway.

Answer 6

The medial myotome becomes the muscles of the back.

1. The dorsal somite becomes the medial dermomyotome which express Pax3 TFs.
2. Wnt signalling from the spinal cord along with Shh signalling from the floor plate and notochord control the expression of Myf5 specifying the cells to be come myoblasts.
3. Myf5 is activated, leading to differentiation of the myoblasts to form myotubules, characterised by the expression of Myogenin and Mrf4.
4. The mytotubules mature to become myofibrils

Question 7

Explain how the hypaxial myotome is specified, differentiated and patterned into body wall muscle. in the embryo. Draw the pathway.

Answer 7

The hypaxial myotome (express Pax3) becomes the muscles of the body wall.

1. Activating Wnt signalling from the ectoderm and inhibiting BMP4 signalling from the lateral plate mesoderm is controlled by MyoD. Noggin, a BMP inhibitor, supresses the BMP inhibition.
2. The MyoD+ specified myoblasts are differentiated to myotubules which express Myogenin and Mrf4.
3. The myotubules mature to become myofibrils.

Question 8

Explain how the hypaxial myotome is specified, differentiated and patterned into limb muscles. in the embryo. Draw the pathway.

Answer 8

The hypaxial myotome (express Pax3) becomes the muscles of the limb.

1. Delamination of the Pax3+ cells from the ventral dermomyotome depends on the presence of c-Met, a hepatocyte growth factor receptor (tyrosine kinase) which interacts with HGF in the limb mesenchyme to delineate the route. Lbx1 also helps but we’re not sure how yet
2. Activating Wnt signalling from the ectoderm and inhibiting BMP4 signalling from the lateral plate mesoderm. BMP4 inhibits the expression of MyoD in the limb mesenchyme
3. At the limb bud, the cells organise into dorsal and ventral limb masses and appear to proliferate, now expressing MyoD and Myf5 (now determined)
4. The MyoD+ specified myoblasts are differentiated to myotubules which express Myogenin and Mrf4.
5. The myotubules mature to become myofibrils.

Question 9

Outline the basic concept of muscle regeneration and the role of satellite stem cells.

Answer 9

Satellite cells originate from somites. The cells are used to feed the growth of muscles. and to regulate muscles after injury caused by exercise, stretch or denervation:

• Injury to muscle causes release of reactive oxygen species (ROS) and hepatocyte growth factor (HGF)
• Satellite cells express cMet => activated as a result of the presence of the ROS and binding of HGF (similar process in embryo)
• Satellite cells express Pax3 (another similarity to embryo)
• Molecular program to repair muscles is similar to embryonic program
• Express Myf5 then MyoD, then Myogenin.
• Once Myogenin is expressed, they fuse together to repair muscle.

The way cells in the embryo are regulated can teach us about how cells are regulated in the adult.

Satellite cells important for regeneration but sometimes they aren’t efficient which leads to disease e.g., dystrophies, cachexia, hyperplasia.