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Development and Myogenesis
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Tissue patterning
Cellular construction of muscle
Myogenesis
Synaptogenesis
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
Embryonic structure
Gastrulation
Alberts & Johnson MBoC 2002
Neurulation
Stage 13
Stage 16
Stage 18
Stage 20
Proliferation of neural plate ectoderm
Formation of neural tube
Schroeder, 1970
Somites
• Segmental mesoderm
– Sclerotome (spinal column)
– Dematome (skin)
– Myotome (body & limb muscles)
• Adjacent to neural tube
Christ & Ordhal, 1995
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
Somite transplantation
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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
(now a dead link)
Spatial mapping of somitemuscle
Lance-Jones, 1988
• Rostral somiteproximal
muscle L1 L2 L3 L4 L5 L6 L7
Spinal Segment
Somite
Cross-section through leg
S27 S28 S29 S30 S31 S32
Sartorius
Muscles
Adductors
Somite 29femotibialis, adductor
Femorotibialis
Iliotrochantericus
Post Iliotibialis
Iliofubularis
• Somites don’t know which
muscle they will become
Somite 32iliotibialis, iliofibularis
Neuronal outgrowth
• Growth cone
– Tubulin-mediated extension
– Actin-mediated extension
Forscher & Smith 1988
Molecular Cell Biology. 4th edition.
Lodish et al
Specificity of neural outgrowth
• By coordinated timing? ie: first to arrive
• By coordinated tracking? ie: M&N follow same path
• Cynthia Lance-Jones & Lynn Landmesser
– Retrograde label
of motorneuron
pools
– Flip neural tube
in developing
chicks (before
limb bud formation)
Neurons know their muscles
Inject dye into sartorius
Look for it in neurons
Normal
T7-L3 flipped
• Neuron’s muscle identity is set/committed
early
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
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
Differentiation and Fusion
Myotube #1: some
contractile protein (grey
circles); nucleus (speckled
blob) and nucleolus (egg
yolk)
Myotube #2: less
contractile protein;
nucleus
Myoblast #1:mostly
unorganized, some
mitochondria (circles)
Undifferentiated cells
(2): potentially
myoblasts or
fibroblasts
Myotube #3: Even
less contractile
protein
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
Myofibrillogenesis
• ECM anchor/nucleation
• β-actin & non-muscle
myosin
• Ruler proteins
• α-actin & muscle
myosin
Dabiri et al., 1997
Myofibrillogenesis cartoon
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 3Hthymidine
Nuclei becoming more dense;
randomly labeled
Secondary myotubes (E18) labeled
on E17 with 3H-thymidine
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
Muscle Spindle lineage
• Encapsulated “intrafusal”
fibers
• Sensory
• Nuclear bag
• Nuclear chain
• Dependent on innervation
by DRG
Milburn, 1973
Innervation
• NMJ proteins uniformly expressed in
myotubes
• Innervation
induces clusters
• Agrin (torpedo
organ axons)
Normal NMJ
Failed NMJ: scattered, light
(low density), unorganized
Synapse elimination
• Stephen Turney & Jeff Lichtman
• GFP/CFP/YFP-mouse
– Confocal microscope
– Neonatal variation in
relative expression
– Photobleaching
• Sterrnomastoid muscle
– Repeated imaging over days
Multiple innervations share endplate
• One GFP and one
CFP axon co-localize
and intermingle on a
single fiber
Synapse elimination
• Polyneural innervation during development
• Pruning after birth (Hebbian)
• Selective laser ablation
Turney & al., 2012
Neuronal competition
• Larger/stronger neuron takes over
• Smaller neuron will fill in if larger neuron is
ablated
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
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