Myogenesis (L12) Flashcards
What are muscles used for?
Movement (simple or coordinated) and posture (standing and sitting). Communication (talking and writing, facial expressions), maintaining body temp (heat released through muscle contraction), breathing (diaphragm)
What is muscle wasting caused by?
Injury, ageing or diseases like Becker or Duchenne muscular dystrophy.
What is the gross structure of skeletal muscle?
Skeletal muscle is made up of individual myofibrils that are bundled into fibres. Fibres are bundled into fascicles which then bundle to make muscles.
What is the general sequence from stem cell to muscle?
Muscles start off as stem cells then undergo specification/determination to become a muscle progenitor known as a myoblast. It then continues to differentiate into myotubes, which then mature to become myofibrils. The fibres are then refined to form slow, fast, or intermediate muscles.
Who first had the idea of myogenesis? How was it discovered?
In 1987, Weintraub published an article of the isolation of the myogenesis determination gene (now known as MyoD) - However, due to the limited tool and knowledge (cell culture etc), it was hard to study. He started using fibroblasts C3H10T1/2 which under certain conditions can give rise to certain cell types including myoblasts (needs to be cultured in the presence of 5Aza to get myoblasts - 5Aza is a demethylating agent. So, he next did a differential screening by using 2 cell lines - fibroblast treated with 5Aza and ones that hadn’t. He extracted the mRNA from each cell line and made it into cDNA- so he had 2 pools of cDNA. Because the original cells are similar, they will have a similar gene expression except for the genes that have been induced by 5Aa (which we know is what causes myoblast formation. SO he subtracted cDNA enriched in muscle-specific genes i using hybridisation (genes in both pools will join together and others will remain as single strands). And screened them using myoblast specific probes to isolate what we now know as MyoD cDNA.
What are the properties of MyoD?
MyoD was introduced into a viral vector and used to infect different types of already differentiated cells e.g. pigment, nerve, fat, liver - found it induced all of them to become myotubes which were capable of becoming fully differentiated myotubes. Therefore, MyoD is sufficient for whole muscle differentiation.
MyoD has 2 very conserved domains - its a bHLH protein so has a basic domain which is responsible for binding to DNA, and a HLH domain, which is responsible for the dimerisation of E12 and E47, which is required for the function of the protein.
Other members of the family with very similar structure are Myf5, Myogenin and MRF4. They act as transcription activators. They for heterodimers with E12 and E47 at the E box (CANNTG)
Where do skeletal muscles originate?
Paraxial mesoderm next to the notochord becomes somites. These somites contain the progenitors for skeletal muscle in their myotome. They also contain the dermatome which are precursors for the dermis and skeletal muscle and the syndotome which are the precursors for tendons. The somite originally appears as a ball of potent epithelial cells - they begin to differentiate asymmetrically due to one side being closer to the neural tube. So overall, skeletal muscles come from the dermamyotome
How do somites develop to become different regions of different tissues?
In the ventral part of the somite, EMT occurs, making sclerotome. The dorsal part remains epithelial and flattens out to make dermomyotome. Some of them become mesenchymal and become sandwiched in between the dermamyotome and sclerotome and become myotome. The ones that don’t become dermatome. 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, the Epaxial (medial) and Hypaxial (lateral)
How can you visualise MRFs? what is their expression pattern?
Use in situ hybridisation to visualise where myogenic regulatory factors are expressed in myoblasts during embryogenesis. the expression pattern is highly conserved throughout species. They are expressed in every somite, developing limb bid, brachial arches (contribute to jaw muscles and tongue, and head - ocular muscles). If you do a stain of transverse sections you can see that staining is restricted to exactly the domains of the somites that contain the muscle progenitor.
What gives good evidence that MRFs control myogenesis?
MRFs are activated at different times during embryonic development, but especially during somite and skeletal muscle formation. So the fact they are expressed in the right place at the right time is very strong evidence that it is this family of genes that controls myogenesis. They are also expressed after birth, which suggests they are used in postnatal muscle development too.
How can you look at the specific effects of MyoD?
TO look at the specific effects of MyoD you can do loss or gain of function mutations. To generate KNs in a mouse, you introduce a construct that will disrupt the gene to cells. You select the correctly transfected cells, colonise them and then use those cells to make chimaeras by implanting the embryo into a surrogate. You can then test the offspring for chimerism and ger line transmission. You then cross the heterozygotes to get KO mice and analyse the phenotypes of the KO offspring.
What was found from targetted inactivation studies of MRFs?
Myf5 KO- Earliest gene to be expressed. Mice are viable, no obvious muscle defects at birth. During embryogenesis, you see delays in myotome formation until the onset of MyoD expression, Mtf5 cells migrate aberrantly into sclerotome and dermatome. MyoD KO- Mice are viable, no obvious muscle defects ar birth. During embryogenesis, there is an increased Myf5 expression in the somites to compensate for the lack of MyoD. There is a slight delay in limb muscle development and a deficit in muscle regeneration in adult mice.
MyoD and Myf5 KO - complete absence of skeletal muscles. No presence of myoblasts. Shows Myf5 or MyoD is required to generate myoblasts - doesn’t matter which one you have, you just need one. Mutual compensation (redundancy)
Myogenin KO- mice die shortly after birth from diaphragm defect. Reduced density of myofibres, replaced by myoblasts. Shows myogenin is required for muscle differentiation.
Explain the signalling pathway controlling muscle gene activation
In Epaxial myotome, all progenitors express Pax3, to drive expression of Myf5 we need signals. Which are Wnt, there is a low Shh from the notochord. In the hypaxial myotome, you need Wnt signalling from ectoderm and inhibition of BMP4 from the lateral plate in some cells. This drive MyoD expression. The cells that are inhibited by BMP4 become limb muscles. The blocking of MyoD expression allows the cells to migrate into the limb. SO there is differential regulation of the same genes.
How does limb myogenesis occur?
In cells that need to migrate - Pax3 drives the expression of cMet which is a receptor for growth factors known as HGF/SF. FGF is expressed in the limb bud and acts as a chemoattractant for cells expressing HGF/SF. During migration, they follow either a dorsal or ventral route whilst proliferating. Only after migration will the progenitors differentiate into myofibres by expressing MyoD etc. Hypaxial muscle cells migrate into the limb. We know this occurs due to a naturally occurring mutation called Splotch mice. They have a deletion of the Pax3 gene and therefore Pax3 function. If you look at expression pattern you can see it is normal in the somites, but you don’t see any expression in limbs because the cells failed to migrate. proof that you need Pax3 to trigger expression of cMet to trigger migration into the limb
What are satellite cells?
In adult muscle, satellite cells act as multipotent stem cells for growth. Satellite cells originate from somites. they are clearly visible in limbs from E17.5. 32% of mouse muscle at birth are satellite cells. 5% of mouse muscle nuclei at the adult stage. They are found under the basal lamina of muscle fibres. They are triggered by injury, denervation, exercise and stretch. The remain quiescent until triggered when they then leave quiescence and become activated. When activated, the genes that expressed are exactly the same,e genes in the same order as the ones that control embryogenic myogenesis. They also self replicate to maintain the stem cell pool.
