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BMS 242 - Principles of Developmental Biology > Myogenesis > Flashcards

Myogenesis Flashcards

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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
Q

What are members of the myoD protein family?

A
  • myoD
  • Myf5
  • Myogenin
  • MRF4
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26
Q

What do the myoD protein family do?

A

Act as transcription factors

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27
Q

Where do the members of the myoD protein family bind to DNA?

A

All bind to the SAME sequence in the promoters/enhancers of DNA:

The E box

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28
Q

What is the sequence of the E box in DNA?

A

CANNTG (where N is ANY nucleotide)

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29
Q

Where does skeletal muscle originate from?

A

The dermamyotome

Which is produced by the somites of the paraxial mesoderm

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30
Q

What do somites originally appear as?

A

A BALL of EPITHELIAL cells

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31
Q

Describe the orientation of the somites

A

Has sides that are differently close to:

  • Ectoderm (dorsally)
  • Notochord/spinal cord (medially)
  • Endoderm (laterally)
  • Lateral mesoderm (ventrally)
32
Q

What is the same/different about the cells in the somite when it is originally a ball of cells?

A

Same:
Each cell is EQUALLY multipotent (can form the same structures)

Different:
Different positional information due to the different surrounding structures

33
Q

What happens shortly after the somite has formed?

A

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
Q

Where are the skeletal muscle progenitors?

A

In the myotome

35
Q

What are the 2 distinct regions of the myotome?

What do these regions of the myotome give rise to?

A

Medially - EPAXIAL myotome

Laterally - HYPAXIAL myotome

Give rise to DISTINCT muscles:

  • Epaxial - deep back muscles
  • Hypaxial - trunk and limb muscles
36
Q

What do the cells that DON’T migrate away from the dermomyotome do?

A

They remain EPITHELIAL and give rise to the DERMATOME

37
Q

What does the syndotome give rise to?

A

The tendons

38
Q

What does the endothelial cells give rise to?

A

Blood vessels

39
Q

So, which part of the somite contains progenitor cells for skeletal muscles?

A

The DORSAL part - forms the dermomyotome

40
Q

What do skeletal muscle progenitors express?

A

Pax3 (A paired-box transcription factor)

41
Q

What do pax-3 positive cells contribute to in the trunk?

A

The myotome

42
Q

Where are myogenic regulatory factors expressed?

What is an example of a MRF?

A

In myoblasts during embryogenesis

Pax3

43
Q

Where is Pax3 expressed?

A
  • EVERY somite
  • Developing LIMB BUD
  • Brachial arch
  • HEAD
44
Q

Why is pax3 expressed in the brachial arch?

A

Cells expressing pax3 migrate from here and contributed to the formation of the jaw muscles

45
Q

Why is pax3 expressed in the head?

A

Occular muscles

46
Q

In the somite, where is pax3 expression restricted to?

A

Domains that give rise to the progenitors for the muscles

47
Q

How can the expression of Pax3 be seen in the embryo?

A

In situ hybridisation

48
Q

Describe the timing of myogenic regulatory factor activation during embryonic development

A
  • 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
Q

What studies are carried out to determine if a gene is sufficient for a certain role expected?

A

Gain-of-function/loss-of-function

50
Q

What is the process of gene targeting?

A

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
Q

How select for cells that have taken up the construct through homologous recombination?

A

Select for antibiotic resistance

52
Q

What occurs in a Myf5 KO?

A

Delay in myotome formation

53
Q

What occurs in a MyoD KO?

A

Slight UP-REGULATION of My5f to compensate

Slight delay in limb muscle formation

54
Q

What is ‘functional redundancy’?

A

Genes that are essential for the survival of the embryo - developed mechanisms that still allow survival if the gene is disrupted

55
Q

What occurs in Myf5/MyoD KOs?

What does this show?

A

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
Q

What happens in a Myogenin KO?

What does this show?

A

Presence of myoblasts
BUT, no presence of myotubes

Shows that myogenin is required DOWNSTREAM - for DIFFERENTIATION

57
Q

What is muscle commitment and differentiation mediated by?

What hierarchy?

A

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
Q

What do all the progenitors of the epaxial and hypaxial muscle express?

What is expressed following Pax3 expression?

A

Pax3

Myf5 and MyoD

59
Q

What drives the expression of Myf5 in the progenitors for epaxial muscles?

A

Shh and Wnt signals from the neural tube and notochord

60
Q

What drives the expression of MyoD in the progenitors for hypaxial muscles?

A
  • Wnt signalling from the ectoderm

- In SOME cells: Inhibition BY BMP4

61
Q

In what hypaxial precursors cells is there inhibition by BMP4?

A

In somites that are NEXT TO the limbs

62
Q

Why must there be inhibition of BMP4 in hypaxial muscle precursors of somites next to the limbs?

A
  • 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
Q

How do the cells migrate into the limb?

A
  • 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
Q

What is c-Met?

A

A receptor for the growth factors HGF and SF

65
Q

How is it known that c-Met is important in migration of the hypaxial muscles cells into the limb?

A

Splotch mouse (natural mutant):

  • Deletion in pax3 gene
  • Loss of Pax3 function
  • No c-Met expression
  • No migration into the limb
66
Q

In the adult muscles, what happens to development?

Why?

A

It doesn’t stop

Continue to grow postnatally

67
Q

When does the maturation of muscles occur?

A

Following innervation

68
Q

What cells support the postnatal growth of skeletal muscles?

A

Satellite cells (skeletal muscle specific stem cells)

69
Q

What do satellite cells do during embryogenesis?

A

Remain quiescent

70
Q

Where are satellite cells positioned?

A

OUTSIDE of the muscle fibre

UNDERNEATH the basal lamina

71
Q

When are satellite cells activated?

A

Following injury, exercise or disease (in the adult)

72
Q

What happens in satellite cells when they become activated?

A

They express the SAME genes in the SAME ORDER as the genes that control embryonic muscle formation

73
Q

What happens when the muscle cells resulting from satellite cells differentiate and mature?

A
  • 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
Q

What is % of muscle cells do satellite cells make up in the embryo?

Adult muscle?

A

32% in embyro

5% in adult muscle

75
Q

What causes weak regeneration of the muscle?

What can this lead to?

A

Cells failing to self-renew or proliferate

Can lead to:

  • Muscular dystrophies
  • Loss of muscle in ageing
  • Loss of muscle due to cancer
76
Q

What causes perturbed regeneration of the muscle?

What can this lead to?

A

Too many stem cells produced - excess renewal/proliferation

Can lead to:

  • Cancer
  • Hyperplasia (formation of too many muscle cells)
77
Q

What can hyperplasia be used to treat?

A

Muscular dystophies

BMS 242 - Principles of Developmental Biology (16 decks)

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