Development and Myogenesis

1
Development and Myogenesis

• Tissue patterning
• Cellular construction of muscle
• Myogenesis
• Synaptogenesis

2
Process outline

• Primary myoblasts migrate from somites to muscle
  pools and fuse
• Axons extend from neural tube to muscle pools
• Secondary myoblasts migrate
• Muscle pools separate
• Pruning of multiple innervation

3
Embryonic structure
4
Gastrulation
Alberts Johnson MBoC 2002
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Neurulation
Stage 20
Stage 13
Stage 16
Stage 18
Proliferation of neural plate ectoderm Formation
of neural tube
Schroeder, 1970
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Somites

• Segmental mesoderm
• Sclerotome (spinal column)
• Dematome (skin)
• Myotome (body limb muscles)
• Adjacent to neural tube

Christ Ordhal, 1995
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Quail-chick chimeras

• Nicole Le Douarin (1969)
• feulgen staining show different chromatin
  struct.
• Cells are similar enough to incorporate and
  develop
• Follow specific cells through development

8
Somite transplantation

• Open quail egg
• Inject India ink
• Dissect somite-glob
• Isolate somites
• Implant in chick
• 5-hrs post-op

Nicole Le Douarin, circa 1987, via
sdbonline.org/archive/dbcinema
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Spatial mapping of somite?muscle
Lance-Jones, 1988

• Rostral somite?proximal muscle

L1
L2
L3
L4
L5
L6
L7
Spinal Segment
Somite
S27
S28
S29
S30
S31
S32
Somite 29?femotibialis, adductor
Muscles

• Somites dont know which muscle they will become

Somite 32?iliotibialis, iliofibularis
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Neuronal outgrowth

• Growth cone
• Tubulin-mediated extension
• Actin-mediated extension

Forscher Smith 1988
Molecular Cell Biology. 4th edition. Lodish et al
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Specificity of neural outgrowth

• By coordinated timing? ie first to arrive
• By coordinated tracking? ie MN follow same path
• Cynthia Lance-Jones Lynn Landmesser
• Retrograde labelof motorneuronpools
• Flip neural tubein developingchicks
  (beforelimb bud formation)

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Neurons know their muscles
Inject dye into sartorius
Look for it in neurons
Normal
T7-L3 flipped

• Neurons muscle identity is set/committed early

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Neuronal guidance

• Pioneer Axons
• Segmentally transposed neurons find their
  original targets
• ECM cues (GAGs, fibronectin)
• Secondary Axons
• Follow pioneers
• Cell contact cues

Molecular Cell Biology. 4th edition. Lodish et al
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Final guidance by muscle-cues

• Lewis al (1981)
• Irradiate half the somites?no muscle formation
• Nerves reach the limbs, but fail their final
  branching

Irradiated
Normal
Silver staining shows neurons and their axons
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Differentiation and Fusion
Myotube 1 some contractile protein (grey
circles) nucleus (speckled blob) and nucleolus
(egg yolk)
Myoblast 1mostly unorganized, some mitochondria
(circles)
Undifferentiated cells (2) potentially myoblasts
or fibroblasts
Myotube 2 less contractile protein nucleus
Myotube 3 Even less contractile protein
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Myotube/myoblast interface
Myoblast disorganized (speckled) cytoplasm.
Mitochondria. Filopods extend completely into
myotube
Two cell membranes in intimate, continuous contact
Myotube regular pattern of dots are contractile
myofibrils
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Myofibrillogenesis

• ECM anchor/nucleation
• ß-actin non-muscle myosin
• Ruler proteins
• a-actin muscle myosin

Dabiri et al., 1997
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Myofibrillogenesis cartoon
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Primary and secondary myotubes
Very closely spaced nuclei, many labeled in
clusters.
Primary myotube (mouse E15) labeled on E14 with
3H-thymidine
Still dense nuclei none labeled
Sparse nuclei many labeled
Primary and secondary myotubes (E15.5) labeled
on E14.5 with 3H-thymidine
Nuclei becoming more dense randomly labeled
Secondary myotubes (E18) labeled on E17 with
3H-thymidine
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Myofiber generations

• Primary
• Migrate and fuse before innervation
• Express myosin independent of nerve
• May be constrained to be slow-twitch
• Secondary
• Dependent on innervation
• Majority of adult muscle mass
• Phenotype from nerve activity

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Muscle Spindle lineage

• Encapsulated intrafusal fibers
• Sensory
• Nuclear bag
• Nuclear chain
• Dependent on innervation by DRG

Milburn, 1973
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Innervation

• NMJ proteins uniformly expressed in myotubes
• Innervationinduces clusters
• Agrin (torpedoorgan axons)

Normal NMJ
Failed NMJ scattered, light (low density),
unorganized
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Synapse elimination

• Stephen Turney Jeff Lichtman
• GFP/CFP/YFP-mouse
• Confocal microscope
• Neonatal variation in relative expression
• Photobleaching
• Sterrnomastoid muscle
• Repeated imaging over days

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Multiple innervations share endplate

• One GFP and one CFP axon co-localize and
  intermingle on a single fiber

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Synapse elimination

• Polyneural innervation during development
• Pruning after birth (Hebbian)
• Selective laser ablation

Turney al., 2012
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Neuronal competition

• Larger/stronger neuron takes over
• Smaller neuron will fill in if larger neuron is
  ablated

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Reserve/Satellite cells

• Not all myoblasts fuse into myotubes (2-5)
• Muscle precursor cells
• Outside cell membrane
• Within basal lamina
• Metabolically quiescent
• Muscle-derived stem cells
• Morphologically identical to MPC, 0.2-2
• Pluripotent

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Summary

• Muscles migrate from somites during development
• Fate determined by diffusible factors from
  outside the somite
• Primary myogenesis is independent of innervation
• Secondary myogenesis requires innervation
• Nerves migrate from neural tube
• Target muscle identified intrinsically
• Individual axons compete for specific muscle
  fibers