Lecture 1 - Master regulators of myogenesis

Question 1

What are the myogenic regulatory genes?

Answer 1

• A family of genes coding for beta-helix-loop-helix transcription factors
• essential for muscle development
• expressed exclusively in cells of the myogenic lineage

Question 2

What are the roles of myoD and myf5 in myogenesis?

Answer 2

myoD and myf5 have overlapping yet distinct roles in myogenesis

Question 3

What is the cell lineage of skeletal muscle formation?

Answer 3

1. Fertilised egg 2. Mesoderm 3. Skeletal muscle

Question 4

What structures does the mesoderm form?

Answer 4

dermis bone skeletal muscle kidney heart blood

Question 5

What structures foes the ectoderm form?

Answer 5

External Neural tissues Epidermis

Question 6

What structures does the endoderm form?

Answer 6

Gut Liver

Question 7

What is the process of development of a mesodermal cell?

Answer 7

1. Mesodermal cell 2. Process of determination to a myogenic progenitor 3. Process of differentiation to a multinucleated myofibre

Question 8

What is the process of determination?

Answer 8

When a cell ‘sets aside a lineage’ and is operationally defined Histologically doesn’t appear to be different

Question 9

How do you test whether a cell has undergone the process of determination?

Answer 9

Embryological studies Take out a patch of cells and place in a new position in the embryo Observe whether it switches fate to align with its environment Or maintain fate

Question 10

What are the features of myogenic progenitor cells?

Answer 10

They are commited to a particular cell lineage (undergone determination and proliferative

Question 11

What are the features of multinucleated myofibres?

Answer 11

Undergone differentiation from being a myogenic progenitor cell Clear histology e.g. striation of contraction apparatus (actin, myosin, troponin)

Question 12

How do somites form (temporally) in a chick embryo?

Answer 12

Pair of somites pinch off every 90 minutes

Question 13

Where is the skeletal muscle in the vertebrate body derived from

Answer 13

All the skeletal muscle in the vertebrate body is derived from the somites

Question 14

What are somites?

Where do they form?

What do they form?

Answer 14

Transient mesodermal structures

Form adjacent to the neural tube

Give rise to: skeletal muscle, vertebrae, ribs, dermis of the skin

Question 15

What happens to the somites after their formation?

Answer 15

Mature into three compartments 1. Dermatome 2. Scleratome 3. Myotome

Question 16

What does the myotome give rise to?

Answer 16

Skeletal muscle

Question 17

What three observations pointed to the presence of a dominant regulator of skeletal myogenesis?

Answer 17

1. Muscle/ liver heterokaryons
2. Coordinate activation of muscle genes
3. Activation of a single loci can convert fibroblasts to myoblasts

Question 18

Detail muscle/liver heterokaryons as the initial observation pointing to there being a dominant regulator of skeletal myogenesis

Answer 18

1. Mouse muscle cells and human liver cells (HepG2) were fused
2. Form heterokaryon (fusion of two different cell types) with nuclei sharing cytoplasm
3. Formed muscle with the human forms of actin, myocin and troponin being activiated

Mouse nuclei able to activate the formation of human contractile cells, therefor there is something dominant in muscle cells over liver cells

Question 19

Detail the ‘coordinate activation of muscle genes’ as the second observation pointing to there being a dominant regulator of skeletal myogenesis

Answer 19

Myoblasts, when determined are still proliferative.

When they differentiate: stop dividing, fuse to form myofibres, coordinately activate contractile protein genes (located all over different chromosomes)

These genes are all activated at the same time, therefore pointing to the presence of a dominant regulator of skeletal myogenesis

Question 20

Detail the observation that the activation of a single loci can convert fibroblasts to myoblasts as the third observation pointing to there being a dominant regulator of skeletal myogenesis

Answer 20

1. Mesodermal fibroblasts (3T3 10T fibroblasts) treated with 5-azacytidine, which hypo-methylates DNA (typically activating genes), become myoblasts 50% of the time (high)
2. The frequency of conversion to the myogenic lineage is consistent with the activation of a single genetic loci

Question 21

What are the functions and activities of the myogenic regulatory genes?

Answer 21

• can convert non-muscle cells into muscle cells in culture
• bind to regulatory sequences (E-boxes) in muscle specific genes
• have auto- and cross- regulatory activity

Question 22

Give examples of the myogenic regulatory genes

Answer 22

• MyoD
• Myf5
• Myogenin
• MRF4

Question 23

How was the single genetic locus (observed by treating mesodermal fibroblasts with 5-azacytadine) identified?

Answer 23

Subtractive DNA library

• Isolate RNA in one cell and another
• Make cDNA library
• hybridise and attach to beads

Everything that is the same will hybridise - identified cDNA unique to myoblasts

• identified myoD
• added into 3T3 10T fibroblasts
• transforms to myoblast

Also demonstrated with other cells (fat/nerve) therefore determined master regulator

Question 24

What techniques are available now to look at the difference in genes expressed in a cell?

Answer 24

• RNA sequencing (bioinformatics and computing to compare)
• Microarray

Question 25

How can you determine where genes are expressed?

Answer 25

In situ hybridisation

Question 26

What did an in situ hybridisation identify about the expression of myoD?

Answer 26

Identified uniquely in the somites in the embryo

Question 27

Why is myoD specifically expressed in somites

Answer 27

As master regulator (able to convert other cells fat/nerve to myoblasts) must only be expressed in cells that are going to be muscle

Question 28

Where do transcription factors bind?

Answer 28

To regulatory sequences (6bp)

Question 29

What is the nature of the auto- and cross- regulation of the myogenic regulatory genes?

Answer 29

MyoD binds to an E-box in its own promoter and can regulate its own expression As can Myf5, myogenin, MRF4 Once myoD is expressed it is kept on and turns on the others in the family

Question 30

What is the process of gene targeting in mice? (Rudnicki protocol)

Answer 30

* relies on homolgous recombination * need a targeting vector with regions homolgous to the chosen gene surrounding the active domain * region between the homolgous ends on the target vector will replace active domain (In TF: BHLH DNA binding domain) with neomycin resistance gene * need thymadine kinase outside region of homology 1. If HR successful get new genotype (w/ no TK) 2. double drug selection: neroR/G418 drug selects for insertion, tk/glancyclovir selects for random insertion 3. Must be in ES cells (plutipostent cells derived from the inner cell mass of the mouse embryo) 4. Need breeding strategy 1. chimeric mouse made by injecting double-drug selected ES cells derived from a brown mouse into host blastocyst of a black mouse 2. Chimeric mouse bred to homozygosity, need to check have insertion in the germline cells

Question 31

What happened when Rudnicki knocked out the mygoenic regulatory gene myoD in mice through gene targeting/

Answer 31

NO PHENOTYPE

Question 32

How can conditional knock outs be formed?

Answer 32

CRE/loxp * need targeting vector with lox p sites surrounding BHLH domain and a neomycin resistance gene * loxP sites are recognised by the CRE recombinase which will excise everything flanked by the loxP sites * neomycin resistance necessary for drug selection for insertion in embyonic stem cells (as HR very rare) * gene is not disrupted until combined with CRE * CRE expression can be controlled by attaching to a tetracyclin response factor or place it under the control of a spacial regulatory factor e.g. promoter for the heart/eye (FRT((sites))/FLP works the same way)

Question 33

What was the result of knocking out the myogenic regulatory genes?

Answer 33

Myf5 -/- * delayed formation of the myotome * normal muscle forms after myoD is expressed (myoD expressed later) * no rib formation MyoD -/- * form normal muscle * show increased levels of myf5 expression Myogenin -/- * have a differentiation defect * reduced skeletal muscle MRF4 -/- * normal muscle * increased levels of myogenin

Question 34

What is shown through the phenotypes shown by knockiong out the myogenic regulatory genes?

Answer 34

These genes have redundancy

Question 35

What was the aim of the Rudnicki paper?

Answer 35

Double knock out of MyoD and Myf5 Are they compensating for each other?

Question 36

How did Rudnicki generate double knock out mice?

Answer 36

1. Crossed myoD-/- mice with myf5+/- mice to get mice heterozygous for both myoD and myf5, then crossed these mice 2. At weaning there were no myf5-/- offspring (lack of ribs); no myoD-/- offspring with or without Myf5 3. When delivered by C-section at term all of the genotypes were present 4. Determined by northern blot (mRNA/RNA expression) if the myogenic genes present in different genotypes 1. make radioactive probes for the genes neccessary for muscle development (skeletal actin) (and PGK-1 as control) 2. look for myogenic gene expression in double knock out

Question 37

What was the result of the northern blot showing the expression of myogenic RNA from myoD-/- and myf5-/- knockout mice? (Rudnucki)

Answer 37

* myoD (-/-), myf5 (-/+): reduced differentiation * myoD(+/-), myf5 (-/-): not reduced differentiation * Double knock out: No myogenic genes

Question 38

Aside from the northern blot, how else did Rudnicki demonstrate the result of the myoD/myf5 double knockout?

Answer 38

**Immunohistochemistry (looking at proteins with an antibody)** * double (+/-) mouse lots of alpha-actin and desmin * double (-/-) mouse no alpha-actin or desmin (RNA or protein/northern blot, immunohistochemistry) **Histology** * section through diaphragm and intercostal muscles * double (+/-) lots of muscle * double (-/-) no muscle, few cells (also lost precursor cells). Has lost differentiation and myogenic precursors, therefore the genes are needed very early on in the pathway

Question 39

What is the result of a myogenic K/O? (Hasty 1993)

Answer 39

Myogenin knockout mice do have muscle precursor cells but these fail to differentiate (demonstrated by histology of the tongue) Myogenin knockout mice do not express contractile protein genes but they do express MyoD (northern blots) still present in (-/-) Therefore myogenin is downstream of myoD - needed for differentiation

Question 40

What were Rudnicki and Hasty's conclusions?

Answer 40

Rudnicki: MyoD and Myf5 have overlapping redundent roles in myogenesis in the determination of myogenic progenitors (muscle cells and precursors lost) Hasty: Myogenin has a downstream role necessary for the differentiation of myogenic progenitors (have myogenic precursors but lost skeletal muscle) Thought at the time that MRF4 most highly expressed gene in adults

Question 41

What was the aim of Kassar-Duchossoy (2004)?

Answer 41

To investigate the model of the hierachy (epistasis) of myoD, MRF5, myogenin, mrf4 Made alleles of myf5 mutants using lacZ to see if mice can't make muscle what else do cells become (chondrocytes and other lineages) Also other targeting vectors: 1. BHLH domain replaced with lacZ and neomycin resistance gene 2. BHLH domain replaced by GFP and diptheria toxin, smaller insert 3. conditional knockout: BHLH domain not disrupted instead disrupts the activator gene by breeding with a 'deleter mouse' - expresses ubiquitous Cre recombinase (excises sequences between loxP sites). Following HRdd selection and insertion cassette are removed, smallest construct **All three should have been nulls in myf5** but had different phenotypes

Question 42

What was the result of the three different myf5 null mutants? (Kassar-Duchossoy 2004)

Answer 42

All mutations should risrupt myf5 but don't. H: because myf5 linked in cis to MRF4 the big insertions have an effect Proof: 1. In WT, normal expression of myf5 and MRF4 are expressed strongly before myoD in all somites (in situ hybridisation) 2. In situ hybridisation for Mrf4 in lacZ heterozygote, homozygote and loxP homozygote, Mrf4 expression is only lost in the lacZ homozygote 3. In situ hybridisation for Myf4 or MyoD or Myogenin in the loxP and GFP homozygote, get robust expression of all mrf genes in LoxP homozygote, with decreased expression of genes GFP homozygote 4. Immunohistochemistry of Myf5 loxP MyoD knockout get muscle 5. In situ hybridisation for MyoD in WT, LacZ, loxP and loxP mrf4 knockout, get expression of myoD in WT and loxP. Suggests LacZ also knocks out mrf4 and mrf4 and myf5 neccessary for myoD expression. Explanation: * loxP allele, cleanest * GFP allele, smallest insertion * An allelic serioes, more stronger phenotpyes from different mutations depending on the size of the insert **because genes are linked, the bigger the insertion the stronger the inhibition in cis**

Question 43

How did the Kassar-Duchossoy 2004 paper impact the results from the Rudnicki paper?

Answer 43

Rudnicki paper had big neomycin resistance insertions This affected the expression of the closely linked MRF4 when myf5 was removed, MRF4 also removed. Triple K/O (myoD, MRF4, my5) not double! **Repeated Rudnicki:** Immunohistochemistry: With a clean incision myf5 (-/-) and myoD (-/-) there is RNA and there is muscle

Question 44

What did Hadler investigate? (2008)

Answer 44

MyoD (-/-) mice make normal muscle but show increased levels of myf5 expression What is the nature of the redundancy of myoD and myf5 - are they the same lineage or parallel lineages? Cellular or genetic redudancy

Question 45

What was the structure of the Myf5 transcript as used by Hadler et al 2008?

Answer 45

Insetered CRE recombinase gene anad Neomycin resitance genes (Neo- boardered by FRT sites for FLP recombinase(bad expression if keep in)) into the 3'UTR following the myf5 coding region Added an internal ribosome entry site upstream of CRE so that myf5 and cre transcribed together. Expression of CRE recombinase in all cells that express myf5

Question 46

What was the breeding stratey used by Haldar to specifically label or specifically kill the myf5 expressing lineage? What were the results from this cross?

Answer 46

3 different mouse lines were bred with myf5-NN (no neo-) mice 1. ROSA LacZ: when myf5-NN bred to this mouse the CRE removes the stop cassette upstream of lacZ and lacZ is expressed in all cells expressing myf5 2. ZEG same way, but mice conditionally express GFP 3. ROSA-DTA: mice conditionally express di[theria toxin so when myf5-NN bred to the mouse, DTA is expressed in all cells which express myf5 then they undergo DTA-induced apoptosis Results: 1. Observed myocin heavy chain, bgalactosidase, DAPI in the Myf5-NN-LacZ tissue. Only % of total myonuclei (blue, DAPI) express myf5. 2. Myf5-ZEG mice were immunostained for myoD and GFP(myf5), some showed expression of both, some the expression of only myoD or myf5 Not all myonuclei from Myf-5 expressing lineage. 3. Tested for cell death. Compare ZEG-Myf5 mice to ZEG/Myf5/DTA mice for GFP expression (counterstained with DAPI, saw GFP expression lost in DTA mice. Compare same mice for GFP expression and MyoD expression, saw loss of GFP(myf5) but still had MyoD expression in the DTA mice. 4. RT-PCR of Myf5-DTA compared to WT looking at MyoD - increase in myoD for Myf5-DTA mice

Question 47

What were the conclusions from the Haldar paper?

Answer 47

Not all myoculei are derived from myf5 expressing myoblasts, therefore there are two lineages of myoblasts. Myf dependent and myf5 independent (probably myod)