Myogenesis Flashcards

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1
Q

Why is understanding skeletal muscle important to learn about?

A
  1. Motor Function
  2. Metabolism (maintains body temp and glucose/fatty acid metabolism)
  3. Respiration (importance of diaphragm)
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2
Q

What are some examples of muscle wasting diseases?

A

Injuries
Ageing
Muscle-degenerating disease (dystrophy)

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3
Q

How are muscles made?

A
  • Differentiation of myoblasts (precursor muscle cells) to muscle fibres (myocytes)
  • initially myoblasts are mononucleated
  • They proliferate and fuse to form multinucleated structures called MYOTUBES
  • Myotubes differentiate into muscles fibres made up of repeating sarcomere units containing actin and myosin
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4
Q

What was a myoblast before it became one?

A

A stem cell/progenitor found in the somite’s with the option to choose many cell fates

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5
Q

What happens along the muscle differentiation pathway?

A

cells are gradually going to lose potency and will acquire new characteristics allowing them to be a muscle cell

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6
Q

What are the effects of 5Aza on fibroblasts and what does this tell us?

A
  • 5Aza is a demethylating agent
  • It blocks the activity of an enzyme that adds methyl groups on to genes, silencing them
  • When added to fibroblasts, some become myoblasts which implies some genes have not been silenced, therefore transcribed and taken a myocyte fate
  • Implies progenitor –> myocyte differentiation is a result of differential gene expression
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7
Q

Following the 5Aza experiment, how did scientists investigate what genes were effected?

A
  1. They took cell types treated/untreated
  2. Extracted mRNA
  3. Converted to cDNA
  4. Found cDNA enriched in muscle specific genes
  5. Screening using myoblast-specific probes (derived from mRNA), fishing genes specific to muscles
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8
Q

How would the 5aza gene experiment have been conducted nowadays?

A

Through a Genome-wide sequence
- isolating RNA and sequencing everything
- more precise and accurate account of genes in fibroblast cell line and in myoblast cell line

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9
Q

What is the next step once you have located muscle-specific genes?

A
  • Take the sequence that has been isolated (e.g. The MYOD: Master regulatory gene for myogenesis)
  • Put it in a viral vector
  • Infect various cell types with vector
  • Can observe reprogrammation of cells becoming muscle cells (e.g. pigment cells, nerve cells fat cells, liver cells all becoming muscle cells)
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10
Q

What does the 4 - member family of of myogenic genes consist of?

A

All TFs called myogenic regulatory factors (MRFs)
MyoD, Myf5, Myogenin, MRF4

  • All recognise the same promoter in genes they regulate named E box
  • Present in a large number of genes essential for myogenic differentiation
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11
Q

What is the relevance of proteins E12 and E47 with members of the myogenic regulatory family?

A

E12 and E47 form dimers with MyoD, Myf5, myogenin and MRF4, facilitating their binding to E boxes and therefore regulation of gene expression
- The myogenic regulatory family belong to a group of basic helix-loop-helix proteins, means it can bind to E12 and E47

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12
Q

How is the D-V axis of the somites essential for muscle progenitor formation?

A
  • Ventral somite undergoes epithelial - mesenchymal transition
  • Dorsal remains epithelial called the dermomyotome and contains skeletal muscle progenitors
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13
Q

How does further specialisation of the dermomyotome occur and what does it lead to?

A
  • Further specialisation along medial-lateral axis (medial closest to neural tube)
  • cells closes will form epaxial dermomyotome which contains precursors for the back muscles
  • Closes to mesoderm will contain precursors for the hypaxial (rest of body) muscles
  • Subset of cells in hypaxial which can migrate and contribute to formation of muscle cells in the limb
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14
Q

What do all the precursor cells express and what does it do?

A

Pax 3
- provides a predestined identity to the cells

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15
Q

Is temporal expression of MRFs consistent of when they are forming?

A
  • Expression is quite early of MRFs
  • First gene to be expressed in the limb = forelimb at days 10.5 , first evidence timestamp of cells leaving the somite and going to the limb
  • So because of this early expression it is telling they play a role
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16
Q

How is a KO experiment typically conducted in a mouse?

A
  • Modify embryonic stem cells to introduce mutations/deletions of the interested gene
  • Reintroduced into blastocyst implanted into surrogate mother
  • Test for germline transmission
  • Analyse offspring of animals
17
Q

What are the results of knocking out Myf5 or MyoD?

A

KO Myf5:
- Earliest to be expressed
- No obvious muscle defect
- Implies its a regulatory gene and other genes can make up for it in gene redundancy

KO MyoD:
- Shows same no obvious muscle defect but a slight delay in limb muscle development

18
Q

What happens if you KO both Myf5 and MyoD?

A
  • No genes fpr functional redundancy
  • Complete absence of skeletal muscles
  • shows you need at least one of these genes for muscle development
19
Q

What happens in a myogenin KO?

A
  • mouse died at birth due to defect in the diaphragm
  • found myoblasts were present but they hadn’t differentiated
  • shows myogenin is important in the differentiation of myoblasts to myocytes
20
Q

From the KO experiments what did scientists start gathering about the myogenic gene hierarchy?

A
  • You need Myf5, MyoD or MRF4 to determine the fate of precursor cells- Pax-3 positive somatic cell (a stem cell)
  • But myogenin is only required further on for myoblast to differentiate into myotube
21
Q

How do the MRF genes get activated in the first place?

A
  • Depends on the precursor location and the interaction with secreted molecules from surrounding tissues
22
Q

What specifies the epaxial and hypaxial muscle lineages?

A

Epaxial:
- Cooperation between Shh and Wnt signals to induce Myf5 and MyoD expression

Hypaxial:
- Wnt signals induce Myf5 and MyoD in cells entering lateral myotome
- BMP4 induces Pax3 and represses Myf5 and MyoD in cells fated to the limb bud

23
Q

Why is combinatorial expression of activating and repressing signals important?

A

Ensures you dont expand the expression domain of each gene to ensure accurate expression

24
Q

What causes cells to migrate in limb myogenesis?

A
  • Pax3/7 drives expression of cMet receptor which is the receptor for HGF
  • HGF is produced in the limb bud and secreted causing migration as they are migrating towards the limb attracted by the growth factor (chemoattraction)
  • While migrating they actively maintain downregulated myogenic programming
  • delay myoD and myf5 in cells that populate the limb because this migration is not compatible with the cells differentiating into muscles
25
Q

What happens once the migrating cells reach the limb?

A
  • split into dorsal and ventral muscles and proliferate
  • then activate Myf5 and MyoD and all the other myogenic programmes
26
Q

How do we know that Pax3/7 are important in limb myogenesis?

A
  • Mutation called splotch which is a Pax3 defect
  • Means no development of somitic muscles and an absolute loss of limb muscles
27
Q

What are satellite cells and what are they important for?

A
  • Muscle specific stem cells
  • Originate from somites and are visible in the limbs
  • Decrease in % through ageing
  • Genetically ablating them leads to DYSFUNCTIONAL MUSCLE REPAIR