Development and Myogenesis

• 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

NeurulationStage 20Stage 13 Stage 16 Stage 18

Schroeder, 1970

Proliferation of neural plate ectodermFormation of neural tube

Somites

Christ & Ordhal, 1995

• Segmental mesoderm– Sclerotome (spinal column)– Dematome (skin)– Myotome (body & limb muscles)

• Adjacent to neural tube

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

• 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 somitemuscle

• Rostral somiteproximal muscle

Somite 29femotibialis, adductor

Somite 32iliotibialis, iliofibularis

L1 L2 L3 L4 L5 L6 L7

S27 S28 S29 S30 S31 S32

Spinal SegmentSomite

Sartorius

Adductors

Femorotibialis

Iliotrochantericus

Post Iliotibialis

Iliofubularis

• Somites don’t know which muscle they will become

Mus

cles

Lance-Jones, 1988Cross-section through leg

Neuronal outgrowth

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

Molecular Cell Biology. 4th edition.Lodish et al

Forscher & Smith 1988

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 motorneuronpools

– Flip neural tubein developingchicks (beforelimb bud formation)

Neurons know their muscles

• Neuron’s muscle identity is set/committed early

Inject dye into sartorius

Look for it in neurons

Normal T7-L3 flipped

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

somitesno muscle formation

• Nerves reach the limbs, but fail their final branching

Irradiated Normal

Silver staining shows neurons and their axons

Differentiation and Fusion

Myotube #2: less contractile protein; nucleus

Myotube #3: Even less contractile protein

Myotube #1: some contractile protein (grey circles); nucleus (speckled blob) and nucleolus (egg yolk)

Myoblast #1:mostly unorganized, some mitochondria (circles)

Undifferentiated cells (2): potentially myoblasts or fibroblasts

Myotube/myoblast interfaceMyoblast : disorganized (speckled) cytoplasm. Mitochondria. Filopods extend completely into myotube

Myotube: regular pattern of dots are contractile myofibrils

Two cell membranes in intimate, continuous contact

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

Primary myotube (mouse E15) labeled on E14 with 3H-thymidine

Primary and secondary myotubes (E15.5) labeled on E14.5 with 3H-thymidine

Secondary myotubes (E18) labeled on E17 with 3H-thymidine

Very closely spaced nuclei, many labeled in clusters.

Still dense nuclei; none labeled

Sparse nuclei; many labeled

Nuclei becoming more dense; randomly labeled

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

• Innervationinduces clusters

• Agrin (torpedoorgan 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