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Cell and Developmental Biology > Mesoderm and Myogenesis > Flashcards

Mesoderm and Myogenesis Flashcards

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1
Q

what are morphogens?

A

substance that can elicit different responses in a cell depending on its concentration

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2
Q

what is the idea of morphogens?

A
  • cell needs positional information
  • have a source of a signal
  • at the source the field of cells is really concentrated
  • forms a gradient and diffuses away from the source
  • diffusion from a source establishes a concentration gradient of a secreted signal
  • sensing the concentration of the signal in the immediate environment tells a cell where it is relative to the signalling source
  • cells are able to sense the local concentration of the signal and respond in different ways depending on concentration
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3
Q

describe the french flag model

A
  • simple pattern of 3 different states
  • source → cells have the potential to respond in 3 different ways
  • the source is at one end → it diffuses into a gradient
  • each cell can read out its position and get its positional value
  • gets to the concentration, senses the local environment and finds its position
  • there are discrete thresholds → above which a cell responds one way and below which the cells responds a different way
  • the concentration gradient can be interpreted in many different ways
  • could generate other patterns with different or more threshold response
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4
Q

why are frog embryos good to study

A

lay their eggs external → developing outside the mother → they’re also relatively large eggs → they all divide at the same time and it’s also quite rapid with no growth

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5
Q

describe the stages the frog embryo goes through

A
  • blastula formation
  • gastrulation
  • phylotypic stage
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6
Q

what is the blastula?

A

fluid filled cavitiy, have cells on the outside - need to be rearranged

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7
Q

what is gastrulation?

A
  • forming the gut
  • cells of the mesoderm move in
  • original cavity disappears and a new cavity is formed (the gut)
  • anus forms first and the mouth forms second
  • driven by the dorsal mesoderm
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8
Q

what are the stages of early development?

A 
Cleavage → no growth, rapid
Blastula → fluid filled cavity forms
Gastrula → cell movements, formation of 3 germ layers
Neurula → Neural tissue forms
Tailbud → phylotypic stage
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9
Q

what are the 3 germ layers?

A
  1. ectoderm
  2. mesoderm
  3. endoderm
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10
Q

what does the ectoderm form?

A

epidermis and CNS

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11
Q

what does the mesoderm form?

A

notochord, dermis, skeleton, muscle, kidney, heart and blood

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12
Q

what does the endoderm form?

A

forms the gut, liver, pancreas and lungs

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13
Q

how are the embryo germ layers organised in amphibian blastula stage?

A 
ectoderm = on the animal pole
endoerm = at the vegetal pole
mesoderm = forms the cells at the equator
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14
Q

describe the the formation of mesoderm in blastula

A
  • marginal zon explants from early blastula embryos only form epidermis
  • marginal zone explant from late blastula embryo for mesoderm
  • early blastula = no mesoderm
  • late blastula = there is mesoderm
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15
Q

what do signals from the vegetal pole induce?

A

mesoderm in animal cap (ectoderm) explants

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16
Q

how did they find out that signals from the vegetal pole induced mesoderm?

A

By labeling the animal cap cells with a fluorescent dye, it was found that all the mesoderm is derived from the animal cap
No mesoderm arises from the vegetal cells
We can conclude that the vegetal cells produce a mesoderm inducing signal
Animal cap gives rise to mesoderm in response to signals form vegetal cells

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17
Q

describe the experiment by Peter Nieuwkoop on signals from the vegetal cap

A

Vegetal cells produced two distinct signals
On signal induced extreme ventral mesoderm (mainly blood and smooth muscle)
One signal induced extreme dorsal mesoderm (mainly notochord)
Small dorsal vegetal (DV) region induces extreme dorsal mesoderm
Large ventral vegetal (VV) region induces extreme ventral mesoderm
These experiments idneitfy DV and VV cells as the source of mesoderm inducing signals
However, they do not induce the full range of mesodermal tissues eg muscle and kidney

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18
Q

what do cell lineages show about the muscle?

A
  • that during normal development most of the skeletal muscle comes from the VMZ but in isolation forms very little
  • combinations most of the muscle and kidney come from VMZ derived cells
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19
Q

what do combinations of late blastula/early gastrula VMZ (ventral marginal zone) and DMZ (Dorsal Marginal Zone) will give rise to?

A

all mesodermal tissue types

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20
Q

what is observation 1?

A

marginal zone cells explaned from late blastula embryos form mesoderm

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21
Q

what experiments support observation 1?

A

Experiment 1: Cells of the vegetal pole can induce the cells of the animal pole to form mesoderm
Experiment 2: Cells of the dorsal vegetal pole induce extreme dorsal mesoderm while ventral cells induce extreme ventral mesoderm. SOme types of mesoderm do not form

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22
Q

what is observation 2?

A

Ventral marginal zone cells explanted from early gastrula embryos do not form much skeletal muscle but in normal development these cells do form skeletal muscle

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23
Q

what experiments support observation 2?

A

l muscle
Experiment 3: Combining DMZ with the VMZ explants from early gastrula embryos results in the formation of the full range of mesoderm derivatives
VMZ cells form muscle, kidney etc in response to the DMZ

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24
Q

what does the three signal model do?

A

Provides a framework to think about the signals important during mesoderm induction and patterning

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25
Q

what is the the three signal model?

A

Blastula Stage
1. DV signal induced DMZ
2. VV signal induces VMZ
Late Blastula/early gastrula
3. DMZ source fi signal that patterns the VMZ
Results in formation of full range of dorsal and ventral mesoderm

26
Q

how does TGF beta signalling affect mesoderm formation?

A
  • cells in the animal hemisphere respond to mesoderm inducing signals
  • forms the basis of mesoderm inducing molecules
  • TGFbeta family members activin and nodal are active in this assay
  • Expose to a low concentration of activin or nodal induce ventral mesoderm
  • A high concentration of activin or nodal induce dorsal mesoderm
  • possible morphogen?
  • Nodal is expressed as a dorsal to ventral gradient in the vegetal cells of blastula stage embryo → has a threshold
27
Q

how many TGF beta ligands are there in the hormone genome?

A
  • over 6-
28
Q

describe transforming growth factor-beta signalling

A
  • TGF beta signal through cell surface serine/threonine kinase receptors
  • The intracellular effectors of the TGF beta signalling pathway are SMAD proteins which are activated by phosphorylation
  • On signalling SMAD proteins translocate to the nucleus where they work with other proteins to activate and repress gene transcription
29
Q

what is the evidence that activin like signals are required for mesoderm induction?

A

Dominant negative from of the activin receptor was made: this mutant receptor lacks the intracellular kinase domain
When expressed in cells the dominant negative activin receptor dimerises with normal endogenous receptors and no signal can be transmitted because no phosphorylation can take place
Al activin and nodal signalling is blocked
When mRNA coding for the mutant receptor is injected into Xenopus embryos all nodal signaling is blocked and the embryos that develop form no mesoderm

30
Q

what is preimplantation genetic diagnosis?

A
  • single cell is removed and tested for gene mutation

- tell us that the reomval of one cell at this point doesnt matter

31
Q

what does cloning tell us?

A

that during development nothing in the genome is lost

32
Q

describe differentiation

A

Fertilised egg is totipotent
Early embryonic cells appear identical and are totipotent/pluripotent
During development cell become more restricted in what they can become
In the end over 200 different cell types differentiate in a mammal
Differential gene expression is the basis for the emergence of differentiated cell types
Cell signalling changes the pattern of gene expression that are required for the emergence of differentiated cell types
Cell lineage determination and differentiation are best understood in the skeletal muscle cell lineage

33
Q

where is skeletal muscle in the body derived from in vertebrates

A

somites

34
Q

what are somites

A

segmented blocks of paraxial mesoderm

35
Q

in what order do somites form?

A

new somites form the anterioer end at regular intervals

36
Q

what is the notochord?

A

a mesoderm derivavtive

37
Q

describe, going from dorsal to ventral, the formation of a frog embryp.

A

DORSAL → notochord, skeletal muscle, kidney, connective tissue, smooth muscle, blood → VENTRAL

38
Q

what occurs during somitogenesis?

A
  • forming somites
  • at first cells are unspecified
  • later = regions of the somites become comitted to forming only certain cell types
39
Q

what are the regions of somites?

A
  • sclertome

- dermamytome

40
Q

what does the sclerotome give rise to?

A

Ribs and Vertebrae

41
Q

describe sclerotomes

A
  • Forms in the ventral medial part of the somite
  • Go through EMT
  • Cells of the clerome undergo mitosis
  • Lose their epithelial structure
  • Become mesenchymal cells
42
Q

where does the dermomyotome form?

A

in the dorsal part of the somite

43
Q

what are the 2 sections of the dermomyotome?

A
  • dermatome

- myotome

44
Q

what does the dermatome give rise to?

A

mesenchymal layer of the dorsal skin (the dermis)

45
Q

what does the myotome give rise to?

A

deep muscle of the back and other muscle cells migrate from the ventral lateral part of the myotome to form the muscles of the body wall

46
Q

where is the skeletal cell lineage specified?

A

in a subset of somite cells

47
Q

what signalling is there when skeletal muscle is forming?

A

Signalling molecules are secreted by nearby structures such as the neural tube, notochord, dorsal ectoderm and lateral plate mesoderm
These signals act together to activate myogenesis in the dorsal medial somite cells
The signals do this by activating the expression of regulatory genes that direct cells down the skeletal muscle lineage

48
Q

describe myogenesis

A

Myoblasts are undifferentiated, proliferative cells within the somite committed to forming skeletal muscle (don’t look anything like a muscle cell)
Myoblasts can be isolated from embryos and kept in culture where they proliferate; but will differentiate as muscle when growth factors are removed
Growth factors in the media allow myoblasts to proliferate
Removal of growth factors allow myoblasts to differentiate

49
Q

what happens when myoblasts differentiate?

A
  1. stop dividing
  2. fuse to form multinucleated myofibres
  3. express contractile protein genes
50
Q

what does it take to make a muscle cell?

A

have to activate a large number of cells

51
Q

what are the observations from myogenesis?

A

when myoblasts stop dividing and fuse they activate the expression of a very large number of different genes all at the same time → points to a common transcription REGULATOR → something to turn all the genes on

52
Q

describe the myogenesi experiment looking a liver cells and mice

A

When a human liver cell is fused with a mouse muscle cell the expression of human → muscle specific gene is detected
This means the human nucleus, that was expressing liver genes, is now expressing liver genes, is now expressing muscle genes
This suggests that there is something in the muscle that can activate transcription of muscle genes
Points to a DOMINANT REGULATORY of myogenesis

53
Q

what can activation of a single loci do?

A

convert fibroblasts to myoblasts

54
Q

descirbe the experiment where mouse fibroblast cell line was treated with 5-azacytidine.

A

treated with a drug that hypomethylates DNA 50% of these cells converted to myoblasts
Hypomethylation removes methyl groups from DNA; these methyl groups are usually found in regions of DNA that are not transcribed and removing them activates gene expression
Methyl groups → are repressive, so if they are removed so things will be activated
The number of fibroblasts that convert to myoblasts (50%) is consistent with the activation of a single locus
Points to a DOMINANT REGULATOR of myogenesis

55
Q

describe the experiment that compares mRNAs present in fibroblasts

A

Comparison of the mRNAs present in the fibroblasts to the mRNAs present in the 5-aza myoblasts
Identified a CDNA capable of converting fibroblasts to myoblasts → it was named MyoD

56
Q

what are MyoD

A

First identified member of a family of bHLH transcription factors that regulate the expression of skeletal muscle genes
Other members → myf5, myogenin and MRF4
Bind specific DNA sequences (called E-boxes) to activate gene expression
bHLH transcription factors coded for my myogenic regulatory genes are collectively referred to as the MRFs (myogenic regulatory factors)
Regulators are expressed very early and specifically in the skeletal muscle cell lineage in all vertebrates

57
Q

what is MyodD expression?

A

MyoD and myf5 are expressed very early in somite cells
Myogenin and mrf4 are expressed later
The MRF genes are expressed exclusively in cells in the myogenic lineage

58
Q

what is the MRF regulated pathway?

A

MyoD and myf5 are muscle determination genes
Proliferating myoblasts express MyoD and Myf5 prior to differentiation
Myogenin regulates myoblast fusion and muscle differentiation
Signals form surrounding tissues activate the expression of MRFs in the early somite

59
Q

describe regulation of myogenesis

A

MRFs are basic helix-loop-helix transcription factors
MRFs heterodimerize with other bHLH transcription factors called E proteins (such as E12)
This dimer binds to an E-box, a regulatory sequence found in the enhancer of targets genes
An E-box consists of a sequence of “CANNTG”
The genes transcriptionally activated by MRFs include contractile protein genes like actin, myosin, troponin as well as MRFs

60
Q

how are MRFs ‘master regulators’ of skeletal muscle?

A

MRFs are able to activate the whole myogenic programme
MRFs are bHLH transcription factors
MRFs activate the expression of skeletal muscle specific genes by binding to E boxes present in their enhance/promoters
MRFs auto and cross regulate MRF expression (by biding E boxes)
MRFs are key regulators of muscle development in all vertebrates
A mouse knockout lacking myoD and myf5 has no skeletal muscle (ie they are essential for muscle development)

Cell and Developmental Biology (4 decks)

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