Lecture 2 - Regulating the regulators

Question 1

How does the timing and localisation of MyoD and myf5 expression vary among species? What does this indicate?

Answer 1

Avians

MyoD: expressed first in the medial part of the somite adjacent to the neural tube

Followed quickly by myf-5 and then myogenin

Mammals

myf5 expressed first in somites at 8dpc

myoD expression follows later at 10dpc

Frogs

myoD and myf5 are activated in the early mesoderm

Mice

all 4 MRF genes have different expression patterns during early development

Distinct expression patterns of the MRFs indicate that each has a distinct role during skeletal muscle development

Question 2

Give an example of the different expression patterns of the MRF genes in mice

Answer 2

myf5 is initially expressed in the dorsal medial (epaxial) mytome very early

myoD expressed ventral lateral (hypaxial) myotome later

Question 3

Why do myf5 (-/-) mice have delayed skeletal muscle formation?

Answer 3

mrf5 is expressed before myoD in mice

Question 4

What do MRFs bind?

Answer 4

MRF are BHLH proteins that bind E-boxes (simple base pair)

Question 5

How do somites form?

Answer 5

Condense from the segmental plate mesoderm as balls of columnar epithelial cells

Happens in the anterior to posterior (rostral to caudal) direction

Question 6

What do the hypaxial cells of the myotome form?

Answer 6

migrate out and form muscles of the limbs

Question 7

What is the hypaxial myotome otherwise known as?

Answer 7

the ventral lateral myotome

Question 8

What is the epaxial myotome otherwise known as?

Answer 8

the dorsal medial myotome

Question 9

How have lineage studies demonstrated the fate of of the medial and lateral halfs of the somites?

Answer 9

Using chick/quail chimeras (quail cells have dense purple nuclei - replaced medial part of the somite with quail somite) of half somite grafts

• the medial part of the early somite gives rise to the myotome and epaxial muscles
• lateral part of the early somite gives rise to cells that migrate to the limb and for hypaxial muscles

When the medial and laterial parts of the early somite are swapped, the somite reorganises and gives rise to normal myotome and limb musculature. Showing the somite cells are still plastic in the early somite and the local environment influences their speciation

Question 10

How did Powell demonstrate visually that there may be an external signal influencing the speciation of the somites?

Answer 10

1. In situ hybridsation
   
   • for MyoD in newly segmented somites: expression is adjacent to the axial structures ) NT/NC
2. Embryological manipulations
   
   • If move the somite, respecifies because of the signals around it

Question 11

What is the environment of the somite?

Answer 11

Medial part of the somite up against the dorsal midline (axial) structures: neural cord tube and notochord

Question 12

How do somites form?

Answer 12

Condense from the segmental plate mesoderm in balls of columnar epithelial cells

In an anterior to posterior direction (rostral caudal)

Question 13

What is an inducer and what is the mesoderm induced by?

Answer 13

Mesoderm: FGF, nodals

Inducing: cell signalling tells cells to be a certain part e.g. mesoderm/ectoderm

Question 14

What did Powell show about MRF expression in the early somite?

Answer 14

Both Myf5 and MyoD on in the early somite

MyoD only expressed in the medial domain

If move the early somite it respecifies because f the signals around it

Question 15

What did Powell demonstrate about the MRF expression in the somites by embryological manipulations?

Answer 15

Used embryological manipulations to demonstrate that the mideline structures (specifically the notochord) are required for the activation and maintenance of MRF gene expression in the somites

Question 16

What was the process of embryological manipulations that Powel used to show that the midline structures (specically the notochord) are required for expression and maintenance of MRF gene expression in the somites?

Answer 16

Embryological manipulations

1. Manipulated the embryo so that somites on one side of the midline structures (neural tube and notochord) were separated from the midline structures - so that no signal coming from the midline structures could reach the somites
2. In the somites that were left next to the midline structures, expression of MyoD is maintained (ISH) but in the case where the somite developed away from the notochord MyoD and Myf5 not activated

PART 2: The notochord is required for the activation of mrf gene expression in the somites

1. Split neural tube so that half somites developed with adjacent to the neural tube alone, and half next to the neural tube and notochord; also removed the notochord: found that only somites that developed adjacent to the notochord showed expression of myoD (although there was somitagenesis without the notochord)

If notochord is removed lose expression of myoD

Question 17

Where is sonic hedgehog produced?

Answer 17

Shh is produced in the floorplate and the notochord

Question 18

How is the extracellular Shh percieved by the responding cells and how was this elucidates?

Answer 18

Mutagenesis screens in drosophila

Identified a number of genes in the hedgehog pathway

• patched (ptc), smoothened (smo) and cubitus interuptis (ci)
• without hh, ptc repressed smo and the cos/fu microtubule complex causes the transcription factor Ci to be processed into the smaller form which repressed hh target genes
• in the presence of hh binding to ptc (transmembrane receptor), ptc is inhibited and smo is activated. Smo interacts with cos/fu complex (group of proteins associated with microtubules to disrupt the assocation with the microtubules), resulting in the stabliisation of the full length Ci which can move to the nucleus and function as a transcriptional activatior of hh target genes

Question 19

How is the hh signalling pathway further maintained when it is active by hh binding to ptc?

Answer 19

ptc is one of the target genes for hh signalling, maintaining hh pathway

Question 20

How do we investigate whether a gene is important for a particular process?

Answer 20

If in mice, do a knockout of the gene

Question 21

What was the purpose of the Borycki study?

Answer 21

Knock out shh in mice and evaluate whether myf5 or myoD expressed is affected in the somites

Question 22

What did Borycki demonstrate about shh null mice?

Answer 22

Neither myf5 nor MyoD are expressed in the epaxial dermamyotome

but are expressed in the hypaxial dermamyotome

Conclusion: Shh needed for the expression of myf5 and MyoD in the epaxial dermatome

Question 23

What critism was there of Borycki’s conclusion that shh is needed for epaxial expression of myf5 and myoD ? How was this critism disproved?

Answer 23

The cells may just be dying as shh is (or was thought) to be necessary for survival

Assay for cell proliferation - BrdU labelling

1. Put uracil into embryos with a modification so that it can be observed. Inject pregnant mothers with BrdU which gets taken up if dividing at the time of injection (both WT and shh (-/-)
2. Mitotically active cells found in the dermamyotome of both WT and shh null mice

Assay for lineage specific apoptosis in the epaxial dermamyotome in shh null mice

1. Used an antibody based assay (TUNEL staining). When cells die, DNA begins to form dsbreaks. TUNEL staining chemically labels the end of broken DNA for the detection of apoptosis in tissue sections
2. Apoptotic cells found in the ventral somite in both WT and shh null mice, not in the dorsal somite
   
   1. Therefore apoptosis does not explain the failure of the epaxial cells to activate myf5 and myoD expression

Question 24

What does TUNEL staining stand for?

Answer 24

Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labelling

Question 25

How did Borycki confirm that shh induces myoD?

Answer 25

Figure 5 (A) Took explant of presomitic mesoderm a) alone (with or without shh) b) with just the surface ectoderm (with or without shh) c) with the neighboring tissue Results a and b) if shh not included, get little myoD expression a and b) when shh added there are some myoD postive cells c) in the presence of the midline structures (so long as the surface ectoderm is also present) there is far higher # of myoD expressing cells

Question 26

What was shown by Borycki by experiemt of Myf5 (-/-) mice?

Answer 26

Looked at different genotypes of mrf5 (-/+) (+/+) (-/-) Take explants from mrf5 and treat with NC and NT or shh In myf5 (-/-) with shh don't get MyoD Need myf5 to turn on MyoD Therefore shh activates mrf5 leading to the expression of myoD **But** there are some MyoD positive cells in the presence of the NC, NT and SE even in myf5 (-/-) **therefore** some other signal than Shh is present in these structures which can act independenetly of myf5 to activate myoD

Question 27

How was it further confirmed by Borycki (figure 5 C) that some other signal than Shh is present in these NC/NT/SE which can act independenetly of myf5 to activate myoD

Answer 27

rt-PCR Shh or NC/NT can activate Myf5 expression Added Shh at different concentrations 200mg/ml and 500 mg/ml and looked at myf5 expression

Question 28

What did the summer bell paper show about the normal expression of myf5?

Answer 28

In situ hybridisation for Myf5 Endogenous normal expression of myf5 progresses in the rostral to caudal direction of somite develoment In the dorsal medial tip of the somite get increasing expression of myf5 the older the somite Amplification of my5 as the somite gets older

Question 29

What was the aim of the summer bell paper?

Answer 29

Define what is neccessary to turn myf5 on in different regions of the embryo

Question 30

What is the process of promoter bashing as used by the Summmer Bell paper?

Answer 30

1. Isolate large piece of genes (14kb) and alter ATG of myf5 so it is attqached to lacZ (turns substrate blue) 2. Use pronuclear injection to get the construct into a mouse embryo 3. Implant into mouse 4. Look at phenotype 5. Cut with restriction enzymes until notice phenoype with loss of target gene in particular region/timescale

Question 31

When does pro-nuclear injection take plant?

Answer 31

When see two pronuclei - if the linjected linear DNA is integrated into one of the chromosomes it will be inherited by all cells. Integration needs to occur before the DNA is copied, prior to first cleavage - inject eith male or female pro-nucleus before fusion.

Question 32

Why use pro-nuclear microinjection over ES cell

Answer 32

Es cells more complicated, need double drug selection For the SUMMER BELL paper: technique not gene targeting, the injected DNA can land anywhere and insert into many places eg. may be silenced, into an important gene (mouse dies)

Question 33

With the first, 14kb lacZ construct Summer Bell used what was the result?

Answer 33

Looked at mouse embryo at ten days, had expression of blue somites as predicted.

Question 34

Aside from the 14kb lacz construct, what other constructs were used for the deletion mapping of myf5?

Answer 34

Used EcoRI and StuI restriction site to cut the construct EcoRI larger construct: mouse looks very similar at 9.5 days to the lacZ constuct StuI smaller construct: Expression lost in epaxial somites, therefore the sequence needed for normal epaxial expression in the cut out region of construct #3 Identified regions containing epaxial promoter/enhancer

Question 35

Following the identifiction of the myf5 epaxial promoter/enhancer region what was done? (summer bell)

Answer 35

Add in that limited region fused to lacZ (485kb) Observe that it restores the epaxial expression of lacZ Also identified an enhancer for the branchial arches - expression also restored

Question 36

Following the restoriation of epaxial somite expression and branchial arch expression by the epaxial and branchial arch enhancers attached to lacZ, what was done? (Summer Bell)

Answer 36

Isolated 140kbs of region around mrf4 and myf5 and repeated previous analysis. Defined the different elements of myf5 expression to determine what part of the myf5 locus determines particular expression. Overall they were measuring the necessity of the NT and NC to express myf5 - shh turns myf5 expression on in the epaxial somites

Question 37

Following the identification of the regions of the myf5 locus that control myf5 expression in the somites and that shh drives the expression of myf5, what was the aim of the summer bell paper?

Answer 37

How does shh turn on the myf5 expression? Needs a particular part of the myf5 DNA, likely to be Gli binding sequence in DNA

Question 38

What did the Gustafsson paper show and how?

Answer 38

Binding site for Gli in Myf5 locus * some levels of BMP signalling: repression of shh signalling * some levels of WNT signalling: expression of noggin, inhibits BMP * high levels of WNT signalling from the surface ectoderm activates myf5 * myf5 turns on myoD, as it is earlier, has to integrate signals into a transcriptional output