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Principles of Developmental Biology > Lecture 12- Myogenesis > Flashcards

Lecture 12- Myogenesis Flashcards

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1
Q

What functions are skeletal muscle required for/regulate?

A
  1. Regulates motor function: simple coordinated movement
  2. Regulates metabolism: maintains body function and glucose/fatty acid metabolism
  3. Essential for respiration due to contributing to the formation of the diaphragm
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2
Q

Outline the basic steps in making a muscle

A
  1. Stem cells undergo specification/determination into
  2. Muscle progenitor cells (myoblast) undergo differentiation and fusion to
  3. Differentiated muscle cells (multinucleated myotubes) undergo maturation into
  4. Myofibers which have a specific structure whereby the cytoplasmic content of the cell if organised into microfibrils
  5. Muscles are formed of bundles of cell types called muscle fibres
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3
Q

What are 2 ways in which methylation can occur?

A
  1. Through the addition of methyl group directly to the DNA (through CpG groups). These methyl groups are recognised by specific proteins which recruit other proteins and leads to the remodelling of chromatin and the formation of heterochromatin.
  2. Directly through the methylation of histones which leads to a more compacted chromatin and silencing of genes
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4
Q

Outline how the MyoD cDNA was experimentally identified

A
  1. Took fibroblast C3H10T1/2 cells treated with 5Aza and untreated C3H10T1/2 cells
  2. Isolated the mRNA from both cell types and converted mRNA into cDNA
  3. The cDNA from the untreated cells had the normal composition of genes normally expressed
  4. The treated cells contained normal cDNA as well as cDNA coding for genes that were responsible for converting fibroblasts into myoblasts
  5. Subtracted the two cDNA populations from each other in order to obtain cDNA enriched in muscle-specific genes
  6. Screened cDNA population using myoblast specific probes and identified a single cDNA called MyoD
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5
Q

What is 5Aza and and what affect does it have on fibroblast cells?

A

5Aza ia a demthylating agent and results in the fibroblast cells becoming myoblasts

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6
Q

How does 5Aza convert fibroblasts into myoblasts?

A
  1. Remove methyl groups on DNA and histones and leads to the release of heterochromatin
  2. Reactivates genes which converts cells into myoblasts
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7
Q

What is the result of placing MyoD gene downstream of an active viral promoter and transfecting the construct into different cell types?

A

Irrespective of the cell type, the cells would be converted into myoblasts and eventually multinucleated myotubes that express muscle specific proteins and muscle specific receptors and membrane molecules

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8
Q

What are members of the bHLH protein family that have similar structures to MyoD?

A

MyoD, Myf5, Myogenin and MRF4

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9
Q

What structures and functions are conserved between all MyoD protein family members?

A
  • Transcription activator
  • Helix loop domain form heterodimers with E12/45
  • Basic domain binds to DNA (E box CANNTG)
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10
Q

What gives rise to skeletal muscle?

A

The mesoderm is subdivided and the myotome and dermatome arises from paraxial mesoderm which ultimately gives rise to skeletal muscle

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11
Q

What are the 2 myotome domains and what do they give rise to?

A
  1. Epaxial (medial) myotome gives rise to expaxial muscle which contributes to back muscle formation
  2. Hypaxial (lateral) myotome contributes to body wall and limb muscles
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12
Q

What do the dermamyotome progenitor cells give rise to?

A

Skeletal muscle for the trunk and limbs

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13
Q

What do skeletal muscle progenitor cells express and what do they contribute to?

A

Express Pax3 which in the trunk, contributes to the myotome

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14
Q

If you suspect a gene plays a role in the formation of muscles during development, when do you expect the genes to be expressed?

A

Immediately prior to the first signals of the muscle formation. Specifically in the cell types that contribute to the formation of muscles

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15
Q

What experimental methods allows you to visulaise the distribution of mRNA within the embryo to confirm if a suspected genes plays a role in muscle formation?

A

In situ hybridisation

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16
Q

Outline the steps of gene targeting in ESCs (an example of gene KO/LoF)

A
  1. Introduce mutations in the gene and electroporate into cultured embryonic stem cells
  2. Selection of cells which are resistant to antibiotic and contain mutated gene
  3. Homologous recombination occurs
  4. Analyse/screen colonies for the presence of the mutated gene
  5. Make chimeras by injected ES cells containing mutated genes into organisms blastocyst
  6. Implant blastocyst into surrogate
  7. Test offspring for chimerism
  8. Test for germline transmission by breeding mice and select animals with specific coat colour
  9. Cross heterozygotes
  10. Analyse offspring for phenotype
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17
Q

What can be observed/concluded from a Myf5 mice KO?

A
  • Mice are viable, no obvious muscle defects at birth
  • Myotome formation is delayed until the onset of MyoD expression
  • Myf5 -/- cells migrate aberrantly into sclerotome and dermatome where normally, they would not be expressed
  • Shows functional redundancy: the KO Myf5 is compensated by the activity of MyoD
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18
Q

What is functional redundancy?

A

The KO of one gene is compensated by the activity of another genes

19
Q

What can be observed/concluded from a MyoD mice KO?

A
  • Mice are viable, no obvious muscle defects at birth
  • During embryogenesis, increased Myf5 expression in somites compensates for lack of MyoD
  • Slight delay in limb muscle development and deficit in muscle regeneration in adult mice
  • Shows functional redundancy: the KO of MyoD is compensated by the activity of Myf5
20
Q

What can be observed/concluded from a MyoD/Myf5 dual mice KO?

A
  • Complete absence of skeletal muscles, no presence of myoblasts
  • Proves functional redundancy
  • Myf5/MyoD are essential for the formation of muscles in the embryo
  • Myf5 or MyoD is required to generate myoblasts
21
Q

What can be observed/concluded from a Myogenin mice KO?

A
  • Mice die shortly after birth from diaphragm defect
  • Reduced density of myofibers replaced by myoblasts
  • The formation of myoblasts was not impaired but the myoblasts were unable to differentiate and form myofibers
  • Myogenin is required for muscle differentiation
22
Q

What MFGs mediate the determination of Pax3 somatic cells to myoblasts?

A

Myf5, MyoD and MRF4

23
Q

What MFG mediate the differentiation of myoblasts to myotube?

A

Myogenin

24
Q

What MFG mediates the maturation of myotube to myofibre?

A

MRF4

25
Q

What signalling pathways control the expression of which MRFs in the epaxial myotome?

A

Combination of Wnt and Shh control the expression of Myf5 and downstream the activation of Myogenin and MRF4

26
Q

How does Wnt and Shh signalling control the expression of MRFs in the epaxial myotome?

A
  1. First need Wnt signalling coming from the spinal cord and Shh from the floor plate and notochord
  2. This combination of signalling leads to Myf5 activation and downstream the activation of Myogenin and MRF4
27
Q

What signalling pathways control the expression of which MRFs in the hypoxial myotome?

A

Requires Wnt signalling from the ectoderm and counteracted by BMP4 signalling from the lateral plate mesoderm and contribute to the expression of MyoD and eventually, Myogenin, MRF4 and MRF5

28
Q

How does Wnt and BMP4 signalling control the expression of MRFs in the hypaxial myotome?

A
  1. Wnt signals from the ectoderm
  2. The negative activity of BMP4 can be blocked by the presence of Noggin from the hypaxial lip
  3. The population of cells that remain in the somite and contribute to the body wall muscle can express MyoD because the expression of Noggin blocks the expression of BMP4
  4. However, the cells that will migrate and contribute to the limb muscles cannot activate MyoD because BMP4 is blocking the expression
  5. Both these factors contribute to the expression of MyoD and eventually the expression of Myogenin, MRF4 and MRF5
29
Q

What are the 5 steps in limb myogenesis?

A
  1. Delamination
  2. Migration
  3. Proliferation
  4. Determination
  5. Differentiation
30
Q

Describe the delamination step in limb myogenesis

A
  1. Limb myogenesis initiates with the delamination of Pax3 cells from the ventral lip of the dermomyotome
  2. In the lateral limb, cells express Pax3 and initiate the expression of a receptor called cMet
  3. cMet is the receptor for a growth factor called HGF/SF
31
Q

Describe the migration step in limb myogenesis

A
  1. HGF/SF is expressed in the limb mesenchyme
  2. The chemoattraction of HGF/SF and cMet that leads to the migration of MPCs into the limb mesenchyme
  3. The cells migrate as Pax3 expressing cells
  4. As the limb mesenchyme is expressing BMP4, the cells cannot activate MyoD
32
Q

Describe the proliferation step in limb myogenesis

A

Once in the limb, MPCs organises themselves into two dorsal and ventral muscle masses and appear to proliferate

33
Q

Describe the determination step in limb myogenesis

A
  1. Subsequent to the proliferation, the cells will activate the expression of Myf5 and MyoD
  2. This leads to the commitment of limb progenitor cells to the myogenic lineage
34
Q

Describe the differentiation step in limb myogenesis

A

Later on the cells undergo differentiation under the control of Myogenin and MRF4.

35
Q

Expression of what is required for the migration of hypoxia muscle cells in the limb?

A

Pax3

36
Q

Briefly describe the structure and function of Pax3

A
  1. Paired domain important for the binding to DNA

2. Homeodomain which can also bind to DNA

37
Q

What is a splotch mouse?

A

A mouse which contains a naturally occurring Pax3 deletion mutation. This prevents the expression of MyoD in the limb so no limb bud forms.

38
Q

What is the role of Pax3 in limb myogenesis?

A

Pax3 is required for the regulation of the transcription of the c-met receptor

C-met drives the migration of the cells into the limb muscle through the HGF/SF chemoattractant mechanism

39
Q

What happens to limb myogenesis in the absence of Pax3?

A

In the absence of Pax3, there is no c-met transcription taking place in the somite, no chemoattraction and the cells remain in the somite instead of migrating into the limb

40
Q

Where do satellite cells originate from and what are they required for?

A

Originate from somites during embryonic development and required for muscle regeneration

41
Q

Describe the adult regeneration of skeletal muscle programme

A
  1. Upon muscle injury or exercise, Ros is released which stimulates the release of HGF
  2. HGF binds to c-met on the satellite cells and activates them which leads to them expressing Pax3
  3. MRFs (Myf5 and MyoD) become switched on and are expressed, eventually leading to the expression of Myogenin
  4. They then asymmetrically divide to produce a daughter cell committed for self-renewal and a transit amplifying cell that will expand through symmetric division and finally differentiate
42
Q

What intrinsic and extrinsic cues govern the adult regeneration of skeletal muscle?

A

Notch signaling controls asymmetric division and Wnt signaling is essential in the early phase of satellite cell activation

43
Q

What 5 steps occurs following the activation of satellite cells?

A

1- induction of Myf5 or MyoD
2- expression of both Myf5 and MyoD
3- proliferation/self-renewal
4- differentiation and fusion to existing fibers

44
Q

What diseases can occur from weak or perturbed regeneration of satellite cells?

A

Weak regeneration underpins the progression of muscle dystrophies, sarcopenia and cachexia and can be caused by a lack of satellite cells

Perturbed regeneration underpins cancer (rhabdomyosarcoma) and hyperplasia and may be due to an excess in satellite cells

Principles of Developmental Biology (16 decks)

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