Lecture 12 Myogenesis

Question 1

Skeletal muscle is a paradigm for what?

Answer 1

Skeletal muscle is a paradigm for the cascade of events which cause pluri-/multipotent cells to adopt a restricted/specialised fate and form a tissue

Question 2

What regulates myogenesis during development to bring about the growth/regeneration of adult muscle?

Answer 2

Myogenic Regulatory Factors (MRF)

Question 3

What 3 things are muscles involved in?

Answer 3

• Motor function (simple or coordinated movement that allows standing position)
• Metabolism (maintaining body temperature through glucose/fatty acid metabolism)
• Respiration (diaphragm)

Question 4

Give examples of muscle wasting diseases

Answer 4

• Injuries
• Ageing
• Muscle-degenerating diseases/dystrophies (Duchenne and Becker) – early onset, progressive disease

Question 5

Name the 3 steps in which a stem cell gradually becomes restricted to form myofibers

Answer 5

• Specification/determination of stem cells to form myoblasts
• Differentiation – mononucleate myoblasts fuse to form myotubes
• Maturation to form myofibers

Question 6

What does 5Aza do?

Answer 6

Demethylating agent

It causes cells to differentiate into myoblasts suggesting 5Aza can convert the fate of a cell.

Methylation silences the genome so demethylation will make it more accessible.

5Aza demethylates CpGs upstream of the gene, initiating a cascade of events which decompact the chromatin. Genes normally silenced can now be transcribed

Question 7

What did Weintraub generate and why?

Answer 7

Weintraub generated a differential screen, extracting mRNA from treated and untreated fibroblast, converting both strains to cDNA (containing all the genes specific to fibroblasts, housekeeping genes and new genes converting cells into fibroblasts).

Subtracting the genes present in both removes the housekeeping genes, leaving only the muscle-specific genes which are upregulated or downregulated in the treated cells.

Question 8

What was used to screen the cDNA of the muscle-specific genes and what did it identify?

Answer 8

Myoblast-specific probes

Identify MyoD (myogenic determination gene).

Question 9

Why is MyoD described as a master regulatory gene?

Answer 9

The single introduction of a gene can reprogram differentiated cells into forming muscles, implying this gene has the power to drive the entire myogenic programme. Therefore this gene can drive the entire myogenic programme.

Question 10

How is MyoD exploited?

Answer 10

It can be introduced into an active viral promotor which can then be introduced into a wide range of cell types to drive the conversion into a myoblast cell type which is capable of fusing and forming myotubes

Question 11

Which cells can be dedifferentiated and then change their fate to form muscle with MyoD?

Answer 11

pigment cells, nerve cells, fat cells, fibroblasts, and liver cells

Question 12

What are members of the bHLH family

Answer 12

• MyoD
• Myf5
• Myogenin
• MRF4

Question 13

What happens when the MyoD family proteins are expressed?

Answer 13

All can convert a cell into a muscle cell when overexpressed, acting as transcription factors for the same promoter/enhancer sequence found upstream of muscle-specific genes: the E box (CANNTG)

Question 14

How can we confirm the roles of MyoD genes in muscle formation?

Answer 14

Check that both the mRNA and the protein are present when muscles form i.e. check expressed at right time and in right place - if they’re not there, they can’t be involved! You check both the mRNA and the protein because having the mRNA does not mean you will have a functional protein.

Question 15

What germ layer does muscle derive from

Answer 15

Mesoderm

Question 16

The lateral plate mesoderm contributes partially to what x2

Answer 16

Limbs

Heart

Question 17

Cells then invaginate from the … and … tissue and locate themselves under the dermomyotome to form ….

Answer 17

medial and lateral tissue

embryonic muscle i.e. myotome.

Question 18

What are the 2 myotome domains?

Answer 18

Epaxial (medial)

Hypaxial (lateral)

Question 19

What does medial myotome contribute to

Answer 19

Deep back

Question 20

What does hypaxial myotome contribute to

Answer 20

Bodyward muscles

Question 21

In the… Pax3-positive cells contribute to…

Answer 21

trunk

myotome

Question 22

What does in situ hybridisation of MRFs show in myoblasts during embryogenesis?

Answer 22

Every site of expression (blue) corresponds to where skeletal muscles are forming i.e. in the somites, epaxial and hypaxial muscles, in the limb bud muscles, hypoglossal chord that leads to the muscles of the jaw and tongue, branchial arch and head muscles that allow eyes to function.

Shows that MRFs are expressed in myoblasts during embryogenesis.

Question 23

What is tightly regulated in MRF expression?

Answer 23

Location - tightly restricted to progenitor cells which give rise to muscle

Timing - begins when the first cells begin to specify muscle

i.e. Genes are expressed at the right time and right place for carrying out a function

Question 24

Why do KO of MyoD or Myf5 have similar phenotypes?

Answer 24

Myf5 and MyOD are functionally redundant and bind to the same EBOX protein. In the absence of one gene, the other will carry out the function. That is why the mice show no obvious muscle defect and are viable.

Question 25

Describe the MRF needed at each step in formation of myofiber

Answer 25

See notes

Question 26

Describe signalling in the medial part of the somite i.e. the epaxial myotome

Answer 26

In the medial part of the somite, the epaxial myotome progenitors all express Pax3. Epaxial myotome is acted upon by Wnt from the dorsal region of the neural tube and low levels of Shh from the floor plate of the neural tube and the notochord.

Question 27

Describe signalling in the lateral part of the somite i.e. the hypaxial myotome

Answer 27

In the lateral part of the somite, the hypaxial myotome progenitors all express Pax 3 and is acted upon by Wnt from the ectoderm. BMP4 from the lateral plate mesoderm, which would prevent muscle differentiation, is inhibited by Noggin secreted from the tip of the somite.

Question 28

Why do limbs develop later than somites?

Answer 28

The limb itself develops later than the somite, so its muscles will develop later than those in the trunk. All limb muscle tissue derives from the somite - some somite cells are prevented from differentiating so that there are some left for the limb on the lower part of the dermomyotome.
These cells undergo EMT and delaminate and migrate into the limb where they proliferate. They do not yet express myogenic regulatory factors; only once they’re in position will they receive the signals to determine and differentiate into muscle limbs.

Question 29

Give 2 examples of paired, homeodomain TF that are important in formation of limb muscles

Answer 29

PAX3

PAX7

Question 30

What mutant is due to deletion in PAX3

Answer 30

In the Splotch naturally occurring mutant, there is a defect in the neural crest i.e. the pigment so they have a white spot on their belly. MyoD expression is normal everywhere, except the limbbud so they develop with no limb muscles. i.e. there is a deletion or loss of function of PAX3 so no leg muscles will be formed. PAX3 is necessary, then, to differentiate the muscle.