Lecture 3 - Muscle Pioneer Cells Flashcards
What are the advantages of using Zebrafish as a model system?
- Embryos develop outside of the mother, easier to do fate mapping
- Fast development
- Translusent (good for Ab staining)
- Good genetic model, lots of mutant lines available
- accessible for drug treatment
What are the two distinct fibre types present in skeletal muscle?
Fast twitch
Slow twitch
What are the embryonic origins of fast and slow twitch muscle?
Fast:
Slow
How was the origin of slow and fast muscle precursor lineages deciphered?
Firstly…
Describe the shh signalling pathway
- The receptor of hedgehod is patched, which sits in a complex in the membrane which repressed smoothened. Hh binding to ptc removes the repression of smoothened
- Smoothened is activated and initiates a signal transduction pathway. This involves Fu Kinase which represses Su(Fu) in a complex with Gli/Ci proteins
- Gli/Ci is no longer processed to the short inactive form and the long form of Gli/Ci moves to the nucleus to act as a transcriptional activator of hedgehog target genes (engrailed and ptc)
What data support the concept that shh is critical for the establishment of the distinct muscle lineages in the zebrafish myotome?
a.
How can you demonstrate the pattern of muscle fibre types in mice
Stain adult mouse tissue e.g. leg, with antibodies specific for different isoforms of myosin heavy chains
What are the contraction patterns of the different types of muscle fibres?
Fast myosin: product fast twich contractions
Slow myosin: slow, sustained contractions
How are the fast and slow muscle fibres physiologically distinct?
- S: slow, sustained contractions; F: fast contractions
- Different metabolic rates
- different metabolism
- different innervations
How does the distribution of the different muscle fibre types differ between mice and zebrafish?
In fish, the different muscle types are separated, not mixed up as they are in the mouse.
In zebrafish, how can the slow and fast muscle fibres be unambiguously distinguished?
By immunolabelling
Where do adaxial cells form?
Adaxial cells for adjacent to the notochord
What do adaxial cells give rise to ?
Adaxial cells give rise to slow muscle fibres
Most slow muscle cells become superficial slow fibres, but a few become muscle pioneer cells
In zebrafish, where is myoD expressed?
MyoD is expressed in adaxial cells
When is myoD expression turned on in zebrafish?
MyoD expression is turned on before somitogensis in adaxial cells and later in more lateral cells as the somites form
What is the neural plate called in fish
Neural keel
What cells are adjacent to the adaxial cells in fish?
Notochord and looser lateral plate cells
How are muscle projenitors distinguished into fast and slow myofribres?
- Adaxial cells form slow muscle
- Some remain medial and form the muscle pioneer cells
- Most migrate radially away from the notochord to form the superficial slow fibres on the most lateral part of the somite
How was the migration of the adaxial cells visualised?
Transverse sections through the posterior trunk were immunolabelled with an antibody to mark slow muscle (adaxial) cells and counter saided with Hoechst to reveal nuclei
How long does the mugration of the adaxial cells takes?
5 hours
Aside from a progression over time, how else might developmental figures be interpreted?
Shown as sections posterior to anterior as Anterior sections are more developmentally advanced
How might slow cells be immunolabelled?
Ab against Prox1 protein
Prox1 (has a homeobox - DNA binding domain) and so will stain in the nucleus of the cell
Or against slow muscle myocin heavy chain - stains in the cytoplasm
Also need to mark all nuclei (DAPI - nuclear chromatin staining)
How can medial fast fibres be labelled?
Immunostaining for engrailed (red)
Need to ensure not also slow muscle heavy chain or Prox1 positive
How can muscle pioneer cells be labelled?
Immunostaining with Engrailed, and Prox1
The engrailed rxpressing cells that are also positive for prox1 are muscle pioneer cells
How can it be shown that most of the adaxial cells migrate laterally and become superficial slow fibres? (whilst few remain in a medial position and become muscle pioneer cells)
immunostaining with
- Prox1 (slow muscle marker - green)
- Engrailed
Those that are Prox1 + alone are superficial slow muscle fibres
Those that are Engrailed expressing and Prox1 positive are muscle pioneer cells
Those that only express engrailed are medial fast fibres
Follow migration of these cells prior to mutagenesis in time or anterior to posterior
Why must muscle pioneer cells stain with Prox1?
Because they come from the slow muscle lineage, but are also expressing engrailed
Those that don’t express Prox1 do not come from the slow muscle lineage
What are fate maps used for?
To define the lineage of cells
Why are zebrafish good for fate mapping?
Embryo develops outside of the mother
Outline zebrafish development
1 hour: 4 cell stage. Early cleavage, occurs on top of the yolk cell
4 hour: (sphere) last blastula stage
6 hour: (shield) gastrulatio, dorsal lip = embryonic shield - cells move down and around(used in the hirsinger paper)
What was the purpose of the Hirsinger study?
Fate mapping the myotome at the mid-gastrula (shield) stage
How did Hirsinger fate map to zebrafish myotome at the mid-gastrula (sheild) stage?
- Label early single cell in the marginal zone is injected with rhodamine dextran which can only get to another cell by passing to daughter cells when the injected cell divides
- Development left to continue from gastula stage to tail bud stage (30hpf)
- Sectioning and immunostaining with F59 antibody (stains slow muscle more intensely than fast muscle to determine fate
- Repeat many times to generate a fate map
- Identified a slow domain (more dorsal) and a fast domain
This way can determine whether a cell has become a slow muscle fibre
What was the aim of the hersinger paper following the fate mapping of the zebrafish myotome at the gastrulation (sheild) stage?
Aim: Determine how flexible these cells are at this stage
How did Hersinger determine how flexible cells of the zebrafish gastrula are?
- Furing gastrula (shield) stages, cells are transplanted from the slow domain into the fast domain
- Determined: Change their fate for both types of muscle fibre
Therefore muscle precursors are not yet commited to a slow or fast fate at this stage
Following the determinatrion that zebrafish myotome cells are still flexible to changes at the gastrula stage, what was the aim of the hersinger stufy?
Aim: when do cells become commited
How did hersinger show the competency to change of myotome sebrafish cells at the 3 somite stage and what critisms were there?
3 somite stage:
- adaxial cells transplanted into the lateral plate mesoderm still give rise to slow muscle even though they have been moved [commited to slow muscle]
- lateral cells (fast domain) still give risse to fast muscle even though they have been moved [commited to fast muscle
Critism: the signals needed to become slow are no longer active at the 3 somite stage
Test: Lateral cells taken from the posterior part of the segmental plate mesoderm (developmentally younger) transplanted at the 3 somite stage into the adaxial domain
- about 50% do change fate and become slow muscle, therefor teh adaxial signals are still present
Following confirmation that cells at the 3 somite stage are not competent to change, what was the aim of the hersinger study?
Aim: determine how cells change/maintain fate
What is the status of the slow muscle omitted mutant zebrafish? What do these mutants demonstrate?
Lacks all hedgehog signalling as lacks smoothened
Therefore has no slow muscle: adaxial cells in smu/smoothened mutant embryos adopt a fast fibre fate, and slow domain cells at the shield stage adopt a fast fibre fate
Conclusions: In the absence of hh signalling, muscle precursors in the slow domain do not migrate and adopt a fast fibre fate, the lateral migration of adaxial cells require hh signalling
How did Hersinger experomentally demonstrate that the lateral migration of adaxial cells requires hedgehog signalling?
- Fate mapping, labelling cells in the slow domain
- adaxial cells in smu/smoothened mutant embryos adopt fast fibre fate
- slow domain cells in smu mutant embryos at the shield stage adopt fast fibre fate
- Graft experiment
- Normal, adaxial cells grafted to a medial position at the 3-somite stage will migrate laterally
- The same cells from the smu mutant do not - behave the same as the wild type medial or lateral cells that have recieved no adaxial signal and distribute randomly
Therefore need hh signalling for specification (stay ff) and for migration out
What are the characteristics of the U-type mutants?
smu mutant phenotype
somites no longer chevron shaped, U shaped
(Not the head, has a different somite lineage)
Mutations in which genes give a U-type phenotype?
- smoothened
- sonic you
- detour
- you too
- chameleon
All part of the hh signalling pathway
What is the loss of the chevron shaped somite due to in U mutants?
Mutations in components of the shh pathway
Loss of horizontal myoseptum
How is a U type phenotype produced in the sonic-you phenotype?
the duntion of shh is disrupted
How is a U type phenotype produced in the chameleon phenotype?
Disp1 protein is required for the release of cholesterol modified Hh proteins from the cells in which they are produced. The Disp1 protein is inactivated in the chameleon mutant
How is a U type phenotype produced in the slow muscle omitted phenotype?
Smoothened is the protein responsibel for ah Hh signal transduction into the cell and is inactivated in slow muscle omitted.
How is a U type phenotype produced in the detour and you-too phenotype?
Two Fli zinc finger transcription factors, homolgous to Ci in Drosophila, are disrupted
(Gli1 in detour, Gli2 in you-too)
Mutations in what genes do these mutants code for?
- Sonic-you (syu)
- Slow muscle omitted (smu)
- You-too (yot)
- Detour (dtr)
- Cyclops (cyc)
- Sonic-you (syu): sonic hedgehod
- Slow muscle omitted (smu): smoothened
- You-too (yot): gli2
- Detour (dtr): gli1
- Cyclops (cyc): Not part of the hedgehog signalling pathway. Nodal protein; zebrafish cyclops mutant lack a floorplate so there is no expression of Tiggywinkle hedgehog (Twhh)
What was the purpose of the first figure in the Wolff paper?
To distinguish the different cell types in the zebrafish myotome; muscle pioneers, superficial slow fibres, medial fast fibres and other fast fibres
Green = shh (notochord and floor plate)
Red = engrailed (medial fast fibre)
Blue = slow fibre (superficial slow fibre mononucleated)
Blue and Red: muscle pioneer
Looked at the myotome as it is a structure that forms many different cell types
What is the purpose and results of the Wolff figure 2 immunohistochemistry?
Compare engrailed and slow muscle myosin between WT and a shh- fish using immunohisotchemistry.
All engrailed expressing cells were lost.
slow muscle myosin greatly reduced
somites no longer chevron shaped
What is the purpose and results of the Wolff figure 2 immunostaining?
WT embryo:
- all slow muscle fibres (including MPs) express Prox-1 (green)
- only muscle pioneer cells express Engrailed as well
- engrailed proteins expressed in medial fast fibres but less than in muscle pioneers
When ptc is knocked down with antisense morpholino oligos
- hh signalling is upregaulted because smo inhibition is relieved in the absence of ptc, and as ptc is not there to bind and attenuate hh ligand diffusion
- results in more MPs, more MFF and more SSFs (inc prox-1, increase in engrailed)
How did Wolff demonstrate the hh acts as a morphogen?
H: hh acts as a morphogen, coming out from the floorplate and patterning in the ventral dorsal direction. If so would expect cells to respond differently to different concentrations of hh.
How?
- Cyclopamine inhibits hedgehog signalling in a dose dependent manner
- measured ptc gene expression (receptor and expression is a fast transcriptional response to the hh signal)
- added different concentrations of cyclopamine
Results [dose]
- 15uM MPs are lost (no cells expressing engrailed AND slowmuscle myosin) therefore they need high concentraion of hh, but MFF still present
- 20uM no Engrailed cells
- 30uM the number of superficial slow fibres are reduced
Results [time of treatment]
- cyclopamine treatment between one-cell and 7hpf resulted in a loss of MPs and no effect on MFF
- cyclopamine treatment after 18hr resulted in a loss of MFF but not MPs
Conclusions: MP cells require high concentraion of hh signalling early in development. MFFs require slightly less hh signalling after SSF have migrated. Shh actys as a morphogen to pattern the zebrafish myotome
What are the benefits of pharmacologically inhibiting a pathway
Can change timing and dose
How does cyclopamine block hh signalling?
by binding to smoothened
What type of knock down are morpholino oligos?
post translational inhibition of gene activity
Aside from using morpholino oligos, what other techniques are there for post transciptional inhibiton of gene activity?
siRNAs
What was the aim of the Hinits 2009 study? What was the result of the MO knockdown and immunostaining?
To test the requirement for myogenesis in fish
Marked somites and slow muscle myocin
MyoD (-/-), myf5(-/-) get muscle but
myf5 (-/-) and myoD (-/-) together get no muscle
Myogenin (-/-) in mice get no muscle but can in culture
Results alongside that of Rudnicki: if have no myf5 and no myoD get no muscle, even in fish have overlapping/redundant functions
How can you demonstrate that a knock down by morpholino oligos has knocked down a gene?
Western blot to look at the protein levels using antibodies
How did Hinits 2009 prove that they had successfully knocked down myogenin, myoD and myf5?
For myoD and myogenin used a western blot with ab staining.
Did not have an appropriate antibody for myf5 -> gene modification by TILING.
What is the process of TILING?
Targeting Induced Local Lesions in Genomes (TILLING)
- mutagenise sperm
- breed with WT
- cryopreserve sperm from F1 individuals and isolate genomic DNA
- PCR amplify myf5 region of a genome and sequence, look for a mutation like an early stop codon
- Look for phenotype of myf5 mutant fish
How did Hinits confirm that the myf5 KD with the TILLING mutant?
myoD MO injection into a myf5+/hu2022 incross showed that 25% of the resulting embryos lacked expression of slow muscle markers
Confirmes that Myf5 and MyoD cooperate to drive adaxial slow myogenesis in zebrafish
Some fast fibres persis in embryos lacking both myf5 and myoD - these are dependent on hh signalling - tested by cyclopamine treatment of embryos from the myf5/hu2022 incross injected with myoD MO (ablates residual myhz1)
Define a morphogen
a morphogen is a signaling molecule that acts directly on cells to produce specific cellular responses depending on its local concentration.
