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BMS 242 - Principles of Developmental Biology > Myogenesis > Flashcards

Myogenesis Flashcards

1
Q

What are 4 reasons for skeletal muscle?

A

1) Movement and posture
2) Communication
3) Maintain body temperature
4) Respiration

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2
Q

How does skeletal muscle help to maintain body temperature?

A

Heat released through muscle contraction participates in the control of body temperature (thermoregulation)

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3
Q

How is muscle important in respiration?

A

Diaphragm

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4
Q

What are muscle wasting diseases?

A

Injuries
Ageing
Muscle-degenerating disease (dystorophy)

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5
Q

What is understanding how muscles form and differentiate a paradigm for?

A

Studying cell differentiation

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6
Q

What are muscles made from?

A

Bundles of muscle fibres

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7
Q

How do you get the defined structure of a muscle fibre?

A

Gradual process of:

  • Specification
  • Determination
  • Differentiation
  • Maturation
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8
Q

What happens in the differentiation step of skeletal muscle formation?

A
  • Fusion between many MYOBLASTS to from a MYOTUBE

- And the coordinated ACTIVATION of skeletal muscle specific genes

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9
Q

What is a myoblast?

A

A muscle progenitor cell

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10
Q

What happens during the maturation of a muscle fibre?

A
  • Innervation of the muscle

- Refinement of the TYPE of muscle fibre (into slow, fast or intermediate)

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11
Q

What is the myogenesis determination gene?

A

myoD

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12
Q

How can myoD be isolated?

A

1) Start with fibroblast cell line
2) Have to groups of the fibroblast cell line: one cultured in the presence of 5Azsa and one untreated
3) Collect mRNA from both cell lines and convert into cDNA - get 2 populations of cDNA
4) Subtract the populations of cDNA using hybridisation
5) Screen single strand using myoblast specific probes

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13
Q

Under certain conditions, what does the fibroblast cell line C3H10T1/2 give rise to?

A

Number of cell types, including MYOBLASTS

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14
Q

What is 5Azsa?

Why is it used to culture fibroblasts?

A

A de-methylating agent

Used to get a high percentage of myoblasts from the fibroblasts (instead of other cell types)

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15
Q

How will the cDNA of the fibroblasts treated with 5Azsa be the same/different to the untreated fibroblasts? Why?

A
  • Genes will be similar as they come from the SAME mother cell
  • BUT, some genes will be different - treated cell line will be PARTIALLY converted to myoblast cell type
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16
Q

When is hybridisation on the populations of cDNA performed?

How are the unique genes in either sample isolated?

A

After denaturing the DNA

  • Identical genes will hybridise together - can be eliminated through filtration
  • Unique genes (present in only ONE population) - will NOT hybridise and will remain as SINGLE strands
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17
Q

What does demethylating cells do?

A

Transform heterochromatin into euchromatin and release chromatin from a silenced state

Allowing the transcription of genes that would normally be silenced

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18
Q

What is heterochromatin?

A

Highly condensed DNA

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19
Q

What is euchromatin?

A

Uncondensed DNA

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20
Q

What did Weintraub do?

What were the results of this?

What did this show?

A
  • Isolated myoD
  • Inserted myoD into a viral vector and into a number of cell types that were already DIFFERENTIATED (eg. pigment cells, nerve cells, fibroblasts)

Results:
- Cells lost their differentiation characteristics and converted into MYOBLASTS

  • That were capable of differentiating and fully forming DIFFERENTIATED myotubes

Showing:
- Introduction of myoD into a DIFFERENTIATED cell is SUFFICIENT to reprograme the cell into skeletal muscle

AND

  • Carry out the WHOLE myogenetic programme (including the expression of genes responsible for contractile proteins)
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21
Q

What type of gene is myoD

A

A MASTER REGULATORY gene

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22
Q

What is the myod protein family characterised by?

A

2 domains:

  • Basic domain
  • Helix-loop-helix
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23
Q

What does the basic domain of the myoD protein do?

A

Binds to DNA

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24
Q

What does the helix-loop-helix domain of the myoD protein do?

What is this required for?

A

Forms DIMERS with the proteins that belong to the E12 or E47 family

Dimerisation is required for the FUNCTION of the protein

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25
What are members of the myoD protein family?
- myoD - Myf5 - Myogenin - MRF4
26
What do the myoD protein family do?
Act as transcription factors
27
Where do the members of the myoD protein family bind to DNA?
All bind to the SAME sequence in the promoters/enhancers of DNA: The E box
28
What is the sequence of the E box in DNA?
CANNTG (where N is ANY nucleotide)
29
Where does skeletal muscle originate from?
The dermamyotome | Which is produced by the somites of the paraxial mesoderm
30
What do somites originally appear as?
A BALL of EPITHELIAL cells
31
Describe the orientation of the somites
Has sides that are differently close to: - Ectoderm (dorsally) - Notochord/spinal cord (medially) - Endoderm (laterally) - Lateral mesoderm (ventrally)
32
What is the same/different about the cells in the somite when it is originally a ball of cells?
Same: Each cell is EQUALLY multipotent (can form the same structures) Different: Different positional information due to the different surrounding structures
33
What happens shortly after the somite has formed?
Series of events: 1) Ventral part of somite - ETM - defining the SCLEROTOME 2) Dorsal side - no ETM, remains epithelial - defining the DERMOMYOTOME 3) Medial and lateral aspects of the dermamyotome - cells undergo ETM and place themselves in between the sclerotome and dermomyotome - forming the MYOTOME 4) Cells also emerge in between the sclerotome and the myotome - forming the SYNDOTOME and cells that will become endothelial cells
34
Where are the skeletal muscle progenitors?
In the myotome
35
What are the 2 distinct regions of the myotome? What do these regions of the myotome give rise to?
Medially - EPAXIAL myotome Laterally - HYPAXIAL myotome Give rise to DISTINCT muscles: - Epaxial - deep back muscles - Hypaxial - trunk and limb muscles
36
What do the cells that DON'T migrate away from the dermomyotome do?
They remain EPITHELIAL and give rise to the DERMATOME
37
What does the syndotome give rise to?
The tendons
38
What does the endothelial cells give rise to?
Blood vessels
39
So, which part of the somite contains progenitor cells for skeletal muscles?
The DORSAL part - forms the dermomyotome
40
What do skeletal muscle progenitors express?
Pax3 (A paired-box transcription factor)
41
What do pax-3 positive cells contribute to in the trunk?
The myotome
42
Where are myogenic regulatory factors expressed? What is an example of a MRF?
In myoblasts during embryogenesis Pax3
43
Where is Pax3 expressed?
- EVERY somite - Developing LIMB BUD - Brachial arch - HEAD
44
Why is pax3 expressed in the brachial arch?
Cells expressing pax3 migrate from here and contributed to the formation of the jaw muscles
45
Why is pax3 expressed in the head?
Occular muscles
46
In the somite, where is pax3 expression restricted to?
Domains that give rise to the progenitors for the muscles
47
How can the expression of Pax3 be seen in the embryo?
In situ hybridisation
48
Describe the timing of myogenic regulatory factor activation during embryonic development
- Differences between when the members of the myoD family members arise - BUT, all members of the family are expressed in the muscle progenitors are the time of skeletal muscle formation
49
What studies are carried out to determine if a gene is sufficient for a certain role expected?
Gain-of-function/loss-of-function
50
What is the process of gene targeting?
1) Introduce construct to disrupt the gene - into ES cells | 2) Culture the cells and select for cells that have taken up the construct by homologous recombination
51
How select for cells that have taken up the construct through homologous recombination?
Select for antibiotic resistance
52
What occurs in a Myf5 KO?
Delay in myotome formation
53
What occurs in a MyoD KO?
Slight UP-REGULATION of My5f to compensate Slight delay in limb muscle formation
54
What is 'functional redundancy'?
Genes that are essential for the survival of the embryo - developed mechanisms that still allow survival if the gene is disrupted
55
What occurs in Myf5/MyoD KOs? What does this show?
Complete ABSENCE of skeletal muscle Complete ABSENCE of myoblasts Shows that at least ONE copy of either Myf5 or MyoD is needed for myoblasts to form
56
What happens in a Myogenin KO? What does this show?
Presence of myoblasts BUT, no presence of myotubes Shows that myogenin is required DOWNSTREAM - for DIFFERENTIATION
57
What is muscle commitment and differentiation mediated by? What hierarchy?
Myogenic regulatory factors Hierarchy: - Myf5, MyoD or MRF4 to specify a Pax3 somatic cell to become a myoblast - Myogenin required for MYOBLAST to differentiate into a MYOTUBE - MRF4 involved in the maturation of a myotube into a myofibre
58
What do all the progenitors of the epaxial and hypaxial muscle express? What is expressed following Pax3 expression?
Pax3 Myf5 and MyoD
59
What drives the expression of Myf5 in the progenitors for epaxial muscles?
Shh and Wnt signals from the neural tube and notochord
60
What drives the expression of MyoD in the progenitors for hypaxial muscles?
- Wnt signalling from the ectoderm | - In SOME cells: Inhibition BY BMP4
61
In what hypaxial precursors cells is there inhibition by BMP4?
In somites that are NEXT TO the limbs
62
Why must there be inhibition of BMP4 in hypaxial muscle precursors of somites next to the limbs?
- Hypaxial precursors are in the lateral part of the somite - Have a cell population which gives rise to the muscles of the LIMB (MIGRATE into the limb) - In order to migrate - differentiation must be DELAYED - BMP4 BLOCKS the expression of MyoD and Myf5 - preventing determination and then differentiation and PROMOTES MIGRATION
63
How do the cells migrate into the limb?
- Pax3 drives the expression of c-Met - c-Met is chemo-attracted to HGF in the LIMB MESENCHYME, triggering the MIGRATION of the cells into the LM - In the LM, the cells divide into DORSAL and VENTRAL routes and PROLIFERATE - Only once the cells have migrated and proliferated, will they express Myf5 and MyoD
64
What is c-Met?
A receptor for the growth factors HGF and SF
65
How is it known that c-Met is important in migration of the hypaxial muscles cells into the limb?
Splotch mouse (natural mutant): - Deletion in pax3 gene - Loss of Pax3 function - No c-Met expression - No migration into the limb
66
In the adult muscles, what happens to development? Why?
It doesn't stop Continue to grow postnatally
67
When does the maturation of muscles occur?
Following innervation
68
What cells support the postnatal growth of skeletal muscles?
Satellite cells (skeletal muscle specific stem cells)
69
What do satellite cells do during embryogenesis?
Remain quiescent
70
Where are satellite cells positioned?
OUTSIDE of the muscle fibre | UNDERNEATH the basal lamina
71
When are satellite cells activated?
Following injury, exercise or disease (in the adult)
72
What happens in satellite cells when they become activated?
They express the SAME genes in the SAME ORDER as the genes that control embryonic muscle formation
73
What happens when the muscle cells resulting from satellite cells differentiate and mature?
- Repair of the muscle fibres | - Subset of cells do not differentiation and return to being quiescent - in order to maintain the stem cell pool
74
What is % of muscle cells do satellite cells make up in the embryo? Adult muscle?
32% in embyro 5% in adult muscle
75
What causes weak regeneration of the muscle? What can this lead to?
Cells failing to self-renew or proliferate Can lead to: - Muscular dystrophies - Loss of muscle in ageing - Loss of muscle due to cancer
76
What causes perturbed regeneration of the muscle? What can this lead to?
Too many stem cells produced - excess renewal/proliferation Can lead to: - Cancer - Hyperplasia (formation of too many muscle cells)
77
What can hyperplasia be used to treat?
Muscular dystophies

BMS 242 - Principles of Developmental Biology (15 decks)

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