lecture 14 Flashcards
Models of cell differentiation 1: Muscle differentiation 1. What is meant by the terms ‘determined’ and ‘differentiation’? 2. What are the four stages of development of striated muscle in culture? 3. What are the roles of Pax3, mrf4, myf5 and myoD during determination and differentiation of vertebrate skeletal muscle? 4. In muscle, what are satellite cells? 5. What types of division do satellite cells undergo? 6. What are some of the factors that affect quiescence, activation and prolifer
What is differentiated cell?
- different cell types express particular sets of genes
- as cells move along a path of differentiation they reduce their potency
- terminal differentiation (unless it becomes cancerous)
How can a cell stay differentiated?
a) not become cancerous
b) not be taken out and turned into another cell type
What is cell differentiation?
- cell types that have a clear-cut identity in the juvenile and adult
- from pluripotent inner cell mass cells, differentiation occurs over several cell generations
- gradually the cells acquire new features and their potency becomes more and more restricted
- a differentiated cell is characterised by specialised proteins it contains
What is it important for a cell to have in order to become differentiated?
- factors that turn ON the genes that it needs to become X and turns OFF the genes that would lead it down a different path
- activators and co-activators must be bound before transcription can occur
- repressors can also bind to prevent transcription
- these must be bound before transcription activation or inhibition can occur
What is an example of experimenting with tissue-specific gene expression? What does this show us?
- elastase gene from mouse (with own control region, usually only synthesised in the pancreas) and growth hormone gene from human (own control region, usually only synthesised in pituitary) are identified and isolated
- DNA construct engineered, containing control region for elastase and the gene for human growth hormone
- fertilised mouse egg is injected with the DNA construct
- human growth hormone is made in the mouse pancreas
- Tissue-specific gene expression is controlled by the regulatory regions of genes: shows the importance of control regions
What gene is critically important for making muscle cells?
Myo D
What are the basics for how you would make a muscle cell from the muscle stem cells?
all cell types have stem cells
- satellite cell — sits in the muscle underneath the membrane, quiescent until required
- myoblasts: stem cells undergoing a stage of proliferation once required
- myotubes: differentiation and fusion of myoblasts to form multinucleated tubes
- formation of myofiber
Why was it postulated that we have stem cells in all tissues?
healing
What is the name of the membrane of a muscle cell?
Sarcolemma
Where is the satellite cell?
anchored in the basal lamina/ECM
What is determination?
- the cell gradually differentiates
- at first, it acquires subtle changes e.g. change in activity of a few or many genes
- but differences between cells may or may not be visible
- but the change in gene activity suggests the cell is well on its way to becoming a specialised cell type
mesoderm progenitor –> myoblast = determination
What is MyoD?
- an example of a single gene that can transform fibroblast cells into muscle cells
- a master regulator
- key controlling gene, found only in muscle progenitor cells and muscle cells
- when transfected into fibroblasts and other non-muscle cell types, will induce muscle differentiation
- activated by Pax 3, then myoD activates myogenin to activate muscle differentiation programme (i.e. actin, myosin II etc…)
How do we get the development of striated muscle in culture?
- dividing myoblasts in a culture of growth factors: cell multiplication
- cell alignment: cell multiplication ceases (have to remove growth factors in order for this to happen), they now have two ends/are bipolar
- cell fusion: appearance of muscle-specific proteins, e.g. myosin, actin, tropomyosin (contractile proteins), creatine phosphate kinase
- muscle fiber: spontaneous contractions begin, fusion requires integrin Beta-1
What is a myoblast?
- derived from mesoderm (somites)
- committed to forming muscle
What are the key features of the development of vertebrate skeletal muscle?
- mesodermal progenitor receives external signals that encourage it to become mesoderm
- e.g. myoD is turned on by pax3
- mrf4 is involved in turning on myoD and myf5, these last two factors on their own can turn cells into muscle cells
- adding myoD by itself will turn on the rest of the programme
- myoD and myf5 are in an autoregulatory loop - auto-activation
- these growth factors turn the progenitors into myoblasts, while inhibiting differentiation into multinuclate myotubes
- turning them off will lead to the production of myogenin, which ivokes/induces the differentiation programme
- this results in the muscle specific genes being turned on (actin, myosin II, tropomyosin (contractile), Creatine PK (enzyme))
- these genes start to be expressed at the multinucleate myotube
only when proliferation ceases does differentiation begin
What happens if we knockout myoD (-/-)?
- still make skeletal muscle (Myf5 compensates, level raised)
What happens if knockout myf5 (-/-)?
- still make skeletal muscle but have short ribs
What happens if knockout both myoD -/- and myf5 -/-?
no skeletal muscle
shows these are determination factors
What happens if we knockout myogenin?
- still get myoblasts
- start to get the multinucleated tube forming
- don’t get the muscle specific genes forming
- tells us that myogenin is downstream in the programme
What are the important proteins that make cells differentiate into muscle cells?
- mrf4, myf5, myogenin — can also induce muscle differentiation in non-muscle cells
- myoD and myf5 expressed in myoblasts, myogenin expressed during muscle differentiation
- once myoD is on, it maintains its own expression by an autoregulatory loop
What are satellite cells?
- stem cells of skeletal muscle
- express pax7
- evidence for role of pax7
a) delete pax7 from satellite cell — poor tissue repair
b) in pax7 -/- mice, there are no satellite cells at birth
What are the two modes of satellite cell division?
Asymmetrical:
- apical-basal division involves up-regulation of myf5 in the cell that becomes a muscle cell and upregulation of Pax7 in the stem cell
Symmetrical:
- planar division — Wnt7a appears to drive planar spindle formation
What are the apical and basal sides of a muscle cell?
- basal side is next to the basal lamina
What are the factors controlling satellite cell quiescence, activation and proliferation in vivo?
Not fully understood but:
- quiescence: sarcolemma electrical activity (silence activity with botulinum toxin or denervation, leads to activation), how? why?
- activation and proliferation: muscle tension, growth and hypertrophy, physical trauma, and muscle diseases — may change response of satellite cells to growth factors
- stem cell niche: a compartment within an organ/tissue that supports self-renewal of stem cells while preventing them from differentiating
What is the satellite stem cell niche?
- satellite cell in close proximity to myofiber and encased in ECM (basal lamina)
- unless activated by muscle injury or other stimuli, remain quiescent
- dependence on niche has led to poor success rate for stem cell therapy
why? - because difficult to isolate and expand in vitro culture
- limited time to correct gene defects
- low numbers obtained from muscle biopsy
- need to improve understanding of niche requirements in order to culture
