Lecture 14 - myogenesis (skeletal muscle formation) Flashcards
(38 cards)
What is MRF?
Myogenic Regulatory Factors
What are skeletal functions?
- Motor function
- Metabolism
- Respiration
What is the skeletal motor function?
Simple or coordinated movement
What is the skeletal metabolism function?
Maintains body temperature, glucose fatty/acid metabolism
What is the skeletal respiration function?
Importance of diaphragm (which is a muscle)
What are muscle wasting diseases?
- injuries
- ageing
- muscle-degenerating disease = dystrophy (Duchenne & Becker)
What are paradigm for studying cell differentiation?
- MRF function in skeletal myogenesis
- Mechanisms of cell differentiation
How is a muscle made?
Stem cells (specification) –> muscle progenitor cells - myoblast (differentiation) –> differentiated muscle cells - Myotubes –> (maturation) Myofibres
Describe muscle formation
Muscles formed of bundles of muscle fibres. Myofibres are special as they are made up of a fusion of multiple cells to form a multi-nucleated cell (muscle fibre).
Cells have an elongated structure that are full of cytoplasm, which is full of proteins, which make up the sarcomere (structural organisation of proteins that consist of contractile proteins - myosin & actin - give a muscle its contractile activity).
What is a myotube?
Special cell type - fusion of precursors - one cell with multiple nuclei. Precursor cell is myoblasts. Multiple myoblasts will form myotube.
What is seen before myoblasts?
Form from cells which are undifferentiated (stem cells/pluripotent cells) - cells that arise in the somite. These cells have the potential to form all the different cell type but become muscle.
Cells have to undergo specification - pluripotent to muscle cell. Specification/determination phase. Then needs to turn on lots of genes to form myotubes - Differentiation. Myotubes become myofibres is maturation.
Describe the isolation of myoD
If you add 5Aza to fibroblasts - they would specify into myoblasts.
5Aza is an analogue to Azactazine - able to incorporate into DNA when it is undergoing replication. When 5Aza is incorporated into DNA, it prevents that piece of DNA from being methylated (blocks an enzyme - DNA methyl transferase). This enzyme acts to maintain methylation of specific regions of DNA, when replication is occurring. DNA can usually be methylated at CpG sites. When drug is added, it means CpG sites can no longer be methylated when DNA is undergoing replication. Methylation usually results in silencing of DNA.
The addition of 5Aza, leads to demethylation - no remodelling into heterochromatic & not get silencing.
There will usually be regions of DNA where there is no expression, however addition of 5Aza leads to expression of other genes. The addition of 5Aza to fibroblasts leads to production of myoblasts.
How was myoD identified?
Took fibroblasts treated with 5Aza & fibroblasts that hadn’t, and extract mRNA. From mRNA, he made cDNA libraries. He subtracted cDNA - leading enriched for muscle-specific genes, only being expressed in the first population of cells (that had 5Aza). Then screened cDNA & identidied myoblast specific genes only present in treated cells - found MyoD gene. MyoD (muscle determining gene).
What does activation of the master regulatory gene (MyoD) lead to?
Took DNA sequence for MyoD gene & cloned it into viral vector (promotor) & infected different cell types - overexpressed MyoD.
This led to muscle production in other tissues, e.g. pigment cells & nerve cells, which became myotubes. Called a master regulatory gene.
What are features of the MyoD protein family?
Structure of bHLH protein:
- basic domain (binding to DNA)
- helix-loop-helix (dimerization with E12 & E47 proteins - which are transcriptional regulators)
Members of the family:
- MyoD, Myf5, Myogenin, MRF4
Function:
- Transcription activator
- From heterodimers with E12 & E47
- Bind to E box: CANNTG
What occurs when proteins bind to E-box?
Promoter of genes associated with muscle differentiation & function - e.g. contractile genes, metabolic genes etc.
Can programme cells to become muscle
What is important for development of the muscle
It must be expressed in the right place, right time & what if loss-of-function leads to loss of structure.
One of the first events after the epithelial somite emerge, EMT occurs (epithelial to mesenchymal transition). In the dorsal part, it remained epithelial. Dorsal part is called dermomyotome. This region contains the precursor for the skeletal muscle.
Skeletal muscle is going to derive from the DORSAL PART OF THE SOMITE.
Further patterning of the dermomyotome occurs. It is divided into medial & lateral regions. Medial part of dermomyotome is going to form epaxial dermomyotome (precursor for epaxial muscles - contribute to back muscles). Lateral part forms hypaxial dermomyotome - contribute to body wall muscles.
At the level of the limb, there is a subset of cells, that come from the hypaxial domain that migrate into limb buds and go on to form muscles of the limb.
How do skeletal muscles originate from the dermomyotome?
- the dermomyotome contain progenitor cells for skeletal muscles of the trunk & limbs.
- skeletal muscle progenitors express the paired-box transcription factor: Pax3/
- in the trunk, Pax3-positive cells contribute to the myotome. There are 2 myotome domains: epaxial (medial) & hypaxial (lateral).
All cells located in dermomyotome should contain all precursors for skeletal muscle & should be expressing genes that contribute to formation of skeletal muscle. All of skeletal muscle progenitors should be expressing gene Pax3 (also thought to be important for the formation of muscle - it provides pre-destined identity - myoblasts).
Where are myogenic regulatory factors expressed?
MRFs are expressed in myoblasts during embryogenesis.
MyoD expression in limbs. As well as hypoglossal cord (forms tongue). Brachial arch - forms jaw muscles.
Where is expression if MyoD seen in somites?
Expression of MyoD only in dorsal part of somites. Expressed in the right subset of cells.
Describe timing of MRFs activation during embryonic development
Somite & limb are separated as they form at different type. Timing of them are slightly different, but expressed at the right time.
Describe gene targeting in ES cells regarding MRFs
Loss of function experiment.
Knockout created by modifying embryonic stem cells - mutation or deletion in gene of interest. Electroporate mutated construct into ES cells. Select cells that have the mutation of interest. Reintroduce these ES cells into a blastocyst. Implant blastocyst into mouse (surrogate).
Outcross the mouse & test offspring to see whether mutation have been inherited. Looking for germline transmission.
Once identified offspring that are carrying mutation - a stable line can be created. Analysis of the offspring for phenotype can occur.
Then made mutations in MRFs
What does a Myf5 knockout lead to?
Mice are viable, no obvious muscle defect at birth.
During embryogenesis, delay in myotome formation until the outset of MyoD expression.
Myf5-/- cells migrate aberrantly into sclerotome & dermatome.
What does a MyoD knockout lead to?
Mice are viable, no obvious muscle defect at birth.
During embryogenesis, increased Myf5 expression in somites compensates for lack of MyoD. Slight delay in limb muscle development, and deficit in muscle regeneration in adult mice.
