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Advanced topics in developmental biology > Lecture 1 - Master regulators of myogenesis > Flashcards

Lecture 1 - Master regulators of myogenesis Flashcards

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1
Q

What are the myogenic regulatory genes?

A
  • A family of genes coding for beta-helix-loop-helix transcription factors
  • essential for muscle development
  • expressed exclusively in cells of the myogenic lineage
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2
Q

What are the roles of myoD and myf5 in myogenesis?

A

myoD and myf5 have overlapping yet distinct roles in myogenesis

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3
Q

What is the cell lineage of skeletal muscle formation?

A
  1. Fertilised egg 2. Mesoderm 3. Skeletal muscle
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4
Q

What structures does the mesoderm form?

A

dermis bone skeletal muscle kidney heart blood

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5
Q

What structures foes the ectoderm form?

A

External Neural tissues Epidermis

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6
Q

What structures does the endoderm form?

A

Gut Liver

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7
Q

What is the process of development of a mesodermal cell?

A
  1. Mesodermal cell 2. Process of determination to a myogenic progenitor 3. Process of differentiation to a multinucleated myofibre
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8
Q

What is the process of determination?

A

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

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9
Q

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

A

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

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10
Q

What are the features of myogenic progenitor cells?

A

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

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11
Q

What are the features of multinucleated myofibres?

A

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

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12
Q

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

A

Pair of somites pinch off every 90 minutes

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13
Q

Where is the skeletal muscle in the vertebrate body derived from

A

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

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14
Q

What are somites?

Where do they form?

What do they form?

A

Transient mesodermal structures

Form adjacent to the neural tube

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

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15
Q

What happens to the somites after their formation?

A

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

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16
Q

What does the myotome give rise to?

A

Skeletal muscle

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17
Q

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

A
  1. Muscle/ liver heterokaryons
  2. Coordinate activation of muscle genes
  3. Activation of a single loci can convert fibroblasts to myoblasts
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18
Q

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

A
  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

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19
Q

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

A

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

20
Q

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

A
  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
21
Q

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

A
  • 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
22
Q

Give examples of the myogenic regulatory genes

A
  • MyoD
  • Myf5
  • Myogenin
  • MRF4
23
Q

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

A

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

24
Q

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

A
  • RNA sequencing (bioinformatics and computing to compare)
  • Microarray
25
Q

How can you determine where genes are expressed?

A

In situ hybridisation

26
Q

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

A

Identified uniquely in the somites in the embryo

27
Q

Why is myoD specifically expressed in somites

A

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

28
Q

Where do transcription factors bind?

A

To regulatory sequences (6bp)

29
Q

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

A

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

30
Q

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

A
  • 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
31
Q

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

A

NO PHENOTYPE

32
Q

How can conditional knock outs be formed?

A

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)

33
Q

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

A

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
34
Q

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

A

These genes have redundancy

35
Q

What was the aim of the Rudnicki paper?

A

Double knock out of MyoD and Myf5

Are they compensating for each other?

36
Q

How did Rudnicki generate double knock out mice?

A
  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
37
Q

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

A
  • myoD (-/-), myf5 (-/+): reduced differentiation
  • myoD(+/-), myf5 (-/-): not reduced differentiation
  • Double knock out: No myogenic genes
38
Q

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

A

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
39
Q

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

A

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

40
Q

What were Rudnicki and Hasty’s conclusions?

A

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

41
Q

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

A

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

42
Q

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

A

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

43
Q

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

A

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

44
Q

What did Hadler investigate? (2008)

A

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

45
Q

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

A

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

46
Q

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?

A

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.

  1. 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.
  2. RT-PCR of Myf5-DTA compared to WT looking at MyoD - increase in myoD for Myf5-DTA mice
47
Q

What were the conclusions from the Haldar paper?

A

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

Advanced topics in developmental biology (10 decks)

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