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

5. Neurulation Stage 13 Stage 16 Stage 18 Stage 20 Proliferation of neural plate ectoderm Formation of neural tube Schroeder, 1970

6. Somites • Segmental mesoderm • Sclerotome (spinal column) • Dematome (skin) • Myotome (body & limb muscles) • Adjacent to neural tube Christ & Ordhal, 1995

7. 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 Chick cells Quail cells

8. Open quail egg Inject India ink Dissect somite-glob Isolate somites Implant in chick 5-hrs post-op Somite transplantation Nicole Le Douarin, circa 1987, via sdbonline.org/archive/dbcinema

9. 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 Sartorius Somite 29femotibialis, adductor Adductors Muscles Femorotibialis Iliotrochantericus Post Iliotibialis Iliofubularis • Somites don’t know which muscle they will become Somite 32iliotibialis, iliofibularis

10. Neuronal outgrowth • Growth cone • Tubulin-mediated extension • Actin-mediated extension Forscher & Smith 1988 Molecular Cell Biology. 4th edition. Lodish et al

11. 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 labelof motorneuronpools • Flip neural tubein developingchicks (beforelimb bud formation)

12. Neurons know their muscles Inject dye into sartorius • Neuron’s muscle identity is set/committed early Look for it in neurons Normal T7-L3 flipped

13. 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

14. 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

15. Differentiation and Fusion 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 #2: less contractile protein; nucleus Myotube #3: Even less contractile protein

16. 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

17. Myofibrillogenesis • ECM anchor/nucleation • β-actin & non-muscle myosin • Ruler proteins • α-actin & muscle myosin Dabiri et al., 1997

18. Myofibrillogenesis cartoon

19. 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

20. 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

21. Muscle Spindle lineage • Encapsulated “intrafusal” fibers • Sensory • Nuclear bag • Nuclear chain • Dependent on innervation by DRG Milburn, 1973

22. Innervation • NMJ proteins uniformly expressed in myotubes • Innervationinduces clusters • Agrin (torpedoorgan axons) Normal NMJ Failed NMJ: scattered, light (low density), unorganized

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

24. Multiple innervations share endplate • One GFP and one CFP axon co-localize and intermingle on a single fiber

25. Synapse elimination • Polyneural innervation during development • Pruning after birth (Hebbian) • Selective laser ablation Turney & al., 2012

26. Neuronal competition • Larger/stronger neuron takes over • Smaller neuron will fill in if larger neuron is ablated

27. 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

28. 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