Myogenesis Flashcards
Why is understanding skeletal muscle important to learn about?
- Motor Function
- Metabolism (maintains body temp and glucose/fatty acid metabolism)
- Respiration (importance of diaphragm)
What are some examples of muscle wasting diseases?
Injuries
Ageing
Muscle-degenerating disease (dystrophy)
How are muscles made?
- 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
What was a myoblast before it became one?
A stem cell/progenitor found in the somite’s with the option to choose many cell fates
What happens along the muscle differentiation pathway?
cells are gradually going to lose potency and will acquire new characteristics allowing them to be a muscle cell
What are the effects of 5Aza on fibroblasts and what does this tell us?
- 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
Following the 5Aza experiment, how did scientists investigate what genes were effected?
- They took cell types treated/untreated
- Extracted mRNA
- Converted to cDNA
- Found cDNA enriched in muscle specific genes
- Screening using myoblast-specific probes (derived from mRNA), fishing genes specific to muscles
How would the 5aza gene experiment have been conducted nowadays?
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
What is the next step once you have located muscle-specific genes?
- 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)
What does the 4 - member family of of myogenic genes consist of?
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
What is the relevance of proteins E12 and E47 with members of the myogenic regulatory family?
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
How is the D-V axis of the somites essential for muscle progenitor formation?
- Ventral somite undergoes epithelial - mesenchymal transition
- Dorsal remains epithelial called the dermomyotome and contains skeletal muscle progenitors
How does further specialisation of the dermomyotome occur and what does it lead to?
- 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
What do all the precursor cells express and what does it do?
Pax 3
- provides a predestined identity to the cells
Is temporal expression of MRFs consistent of when they are forming?
- 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
How is a KO experiment typically conducted in a mouse?
- 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
What are the results of knocking out Myf5 or MyoD?
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
What happens if you KO both Myf5 and MyoD?
- No genes fpr functional redundancy
- Complete absence of skeletal muscles
- shows you need at least one of these genes for muscle development
What happens in a myogenin KO?
- 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
From the KO experiments what did scientists start gathering about the myogenic gene hierarchy?
- 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
How do the MRF genes get activated in the first place?
- Depends on the precursor location and the interaction with secreted molecules from surrounding tissues
What specifies the epaxial and hypaxial muscle lineages?
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
Why is combinatorial expression of activating and repressing signals important?
Ensures you dont expand the expression domain of each gene to ensure accurate expression
What causes cells to migrate in limb myogenesis?
- 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
What happens once the migrating cells reach the limb?
- split into dorsal and ventral muscles and proliferate
- then activate Myf5 and MyoD and all the other myogenic programmes
How do we know that Pax3/7 are important in limb myogenesis?
- Mutation called splotch which is a Pax3 defect
- Means no development of somitic muscles and an absolute loss of limb muscles
What are satellite cells and what are they important for?
- 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
