lec 15 - myogenesis

fxn of skeletal muscles

• motor function

• metabolism (body temp, glucoce/fatty acid metabolism)

• respiration (diaphragm)

muscle wasting diseases

• injuries

• ageing

• dystrophies (duchenne and becker)

muscles have become a paradigm for studying

cell differentiation

what are the unique properties of muscle cells

• long

• multinucleated

• cytoplasm is made of contractile proteins that are arranged in sarcomeres (the actin and myosin ||==—==|| things)

making of a muscle cell - what are the basic stages/steps

stem cell → specification/determination → myoblast (muscle progenitor cell) → differentiation → myotubes (differentiated muscle cells) → maturation → myofibers

specification

cell has begun to adopt specific characteristics, but these changes are not yet stable

• if you put the cell in a different environment, the cell will revert and potentially adopt a new cell fate

differentiation

acquisition of stable characteristics toward a specific cell fate that provide unique function to the cell (ex: contractile proteins arranged in sarcomere)

• if you put the cell in a different environment, it will not revert back

muscle cell maturation

innervation + formation of NMJ

describe the Weintraub experiment

• Weintraub used 5Aza on fibroblasts (connective tissue progenitor) → turned them into myoblasts (muscle progenitor)

  • he hypothesized that the agent cuased a change in gene expression, leading the cell to differentiate into a muscle cell

• he extracted cDNA from the mRNA of 5Aza-treated fibroblasts and untreated fibroblasts

• he hybridized the two cDNA libraries to subtract the genes that were common between them

• identified a single cDNA

what was the mechanism behind the Weintraub experiments

• methylation condenses and silences genes

• 5Aza is a demethylating agent - released and activated silenced MyoD → myoblast differentiation

MyoD is called _____ because _____

the master regulatory gene

• if you use a viral vector to infect any differentiated cell with MyoD, that cell will turn into a muscle cell

the MyoD gene is sufficient to reprogram a differentiated cell into a muscle cell

Myogenic regulatory factor (MRF)

small family of TFs that are capable to induce muscle differentiation in a cell— even one that is already differentiated

what type of proteins are the MRF proteins? what are the function of each domain?

• basic helix-loop-helix (bHLH) proteins

  • basic domain binds to DNA

  • HLH domain dimerizes with E12 and E47 (E box) proteins

what are the genes in the myogenic regulatory factor family

• MyoD

• Myf5

• Myogenin

• MRF4

fxns of the MRF family

• transcription activator

• form heterodimers with E12 or E47

• binds to E box in regulatory sequence: CANNTG

• regulates the coding of contractile protein

where do muscles come from

somites = ball of epithelial cells

but they change their structure as they progress along the AP axis

• ventral somite - epithelial-to-mesenchymal transition

• dorsal somite - stays epithelial, forms a dermyotome

  • dermyotome contains myoblasts

  • myoblasts express paired-box TF Pax3

  • Pax3-positive cells contribue to the myotome (space underneath dermyotome where muscles form)

dermyotome vs myotome

dermyotome = the dorsal part of the somite that remains epithelial and facilitate myoblast formation

myotome = space under the dermyotome where myoblasts form

MRFs are expressed in _____ located in _____ during embryogenesis

differentiating myoblasts

myotomes

what are the myotome domains

epaxial (medial) and hypaxial (lateral)

how to know if a gene is important

widespread and heavily regulated expression of a gene in embryonic development

timing of MRF activation during embryoni develompent corresponds with

when muscles are proliferating and differentiating in the embryo

how to test if MRFs are important in muscle cell differentiation

loss of function studies - disrupt the function of the gene

• is theis gene required for a particular function?

gain of function studies - force the expression of a gene

• is this gene sufficient to drive gene expression?

• ex: the viral vector experiment

how to do a LOF study in a mouse

KO

• genetically disrupt the gene of interest

• introduce this gene to stem cells

• select only the stem cells that retain the mutation

• reintroduce the stem cells into a mouse blastocyst

• reintroduce the blastocyst into a surrogate mother

• select pups for chimerism (more chimerism = more likelihood the pup is mutated)

targeted inactivation studies of MRFs - initial results

Myf5 KO: viable, no defect

MyoD KO: viable, no defect

conclusion: there were already indications that these are important - it could be that KO of one can cause the other gene to take on some of its function

targeted inactivation studies of MRFs - adjusted results

Myf5/MyoD double KO: complete absence of skeletal muscles, no presence of myoblasts

• Myf5 or MyoD is required to generate myoblasts

Myogenin KO: mice die shortly after birth from diaphragm defect; reduced density of myofibers replaced by myoblasts

• Myogenin is required for muscle differentiation

  • skeletal muscle formation stopped progressing after a certain point

updated overview of muscle differentiation steps

1. Pax3-positive somitic cell

2. Determination - Myf5/MyoD/MRF4

3. myoblast

4. Differentiation - Myogenin

5. myotube

6. Maturation - MRF4

7. myofiber

TF expression is controlled by

signaling pathways

TFS are controlled by signaling pathways - Where do the signals come from?

They're produced in the tissue surrounding the somites

What are the tissues that surround the somite?

• Axial mesoderm and neural tube

• Ectoderm

• Lateral mesoderm

• Notochord

The tissues that are around the somite contribute to:

1. The activation of the expression of MyoD and other genes

2. Restricting the domain where these genes are expressed

How to create a precise domain of expression?

Combination of factors that act positively and factors that act to restrict expression

Myf5/MyoD requires ____ to drive expression

Wnts from neural tube + low Shh from notochord

muscles in the limb arise from the ____ in the _____

somite in the trunk

how do you get the hypaxial muscle cells initially in the trunk to end up in the limb?

migration step

limb myogenesis - migration step

• delamination

  • Pax3 expresses c-Met, which is an HGF/SF receptor

    • HGF/SF are the hepatocyte growth factors that drive migration

  • Pax3+ hypaxial muscle cells delaminate (detach) from the epithelium

• migration

  • cells travel into developing limb bud, guided by HGF/SF

  • prevent early differentiation by inhibiting MGFs

• differentiation when they arrive

trunk vs limb hypaxial cell expression timing

limb expression happens 1-2 days after trunk expression bc the migration step has to happen

Pax3

the gene that drives the delamination and migration of hypaxial muscle cells

Pax7

similar to Pax3

Pax3/7 are both part of the ____ TFs

paired domain

paired domain TFs

paired domain allow for DNA binding

Splotch

naturally occuring Pax3 deletion mutation → loss of Pax3 function

Splotch mouse phenotype

loss of limbs - no cell migration

how to specify the epaxial muscle lineage

cooperation between Shh and Wnt signals to induce Myf5 and MyoD expression

how to specify the hypaxial muscle lineage

Wnt signals induce Myf5 and MyoD in cells entering lateral myotome

BMP4 induces Pax3 and represses Myf5/MyoD in cells fated to migrate to the limb bud

what are satellite cells

stem cells that reside in muscles and repair them

• originate from somites

satellite cells compse ___% of mouse muscle nuclei at birth, and ___% of mouse muscle nuclei at adult stage

32

5

satellite cells exhibit Pax___

7

not Pax3

satellite cells are located

under the basal lamina

satellite cells are activated by

stimuli (muscle injury, exercise, etc)

what are the steps taken for satellite cells to repair skeletal muscles

same as myogenesis

1. induction of Myf5 or MyoD

2. expression of both Myf5 and MyoD

3. proliferation/self-renewal

4. differentiation and fusion to existing fibers

satellite cell diseases - weak regeneration

muscular dystrophies

sarcopenia (age-related loss of muscle mass

cachexia (severe loss of muscle and fat)

satellite cell diseases - perturbed regeneration

cancer (rhabdomyosarcoma)

hyperplasia (enlargement of an organ due to increase in cell number)