Somite maturation over time:

1
Class 23 Differentiation of Somites Muscle and
Bone
Somite maturation over time the somite
separates into four regions
14.10
2
G 14.10
3
Cell types within the somite
4
Multiple signals determine derivatives of the
somite
Epaxial myotome makes back muscles Reguires
Wnts from neural tube Shh from notochord
Hypaxial myotome makes limb and body wall
muscles Requires BMP4, FGF from lateral plate
mesoderm Wnts from
overlying ectoderm
Sclerotome makes bone (vertebrae and ribs)
Requires Shh from notochord
14.11
5
How is muscle made? The discovery of a master
regulatory factor for muscle
forelimb
eye
MyoD expression in the E11.5 mouse embryo
hindlimb
somites
6
A master regulatory gene for muscle
differentiation MyoD, bHLH family of
transcription factors
bHLH transc. Factors Show binding domain, etc
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Myogenesis Stages in determination and
differentiation of myoblasts
14.12
8
Myotome (only proliferating)
FGF
no FGF
Myoblast (starting to differentiate)
9
Three additional members of the MyoD family were
found, each with specific roles and different
expression patterns
MyoD expressed in hypaxial (limbs) myotome
Myf-5 expressed in epaxial (back) myotome
MRF-4 expresed as myoblasts differentiate into
myotubes and myofibers
Myogenin expresed as myoblasts differentiate
into myotubes and myofibers
10
Phenotypes of mouse knockout mutants for myogenic
genes
MyoD -/-
Normal muscle (developmental delays)
Normal muscle (developmental delays)
Myf5 -/-
MyoD -/- Myf5 -/-
Paralyzed, no myoblasts or muscle
Myogenin -/-
Myoblasts, but few myotubes
Muscle fibers (but delayed)
MRF4 -/-
11
Regulation of MyoD family members
MyoD family members are only active as
heterodimers with other bHLH proteins
12
There are both positive and negative regulators
of MyoD family members
Negative Id Mist1 MyoR (dont bind DNA)
Positive E proteins (E12 and E47) (bind DNA)
Timing of differentiation is controlled by
levels of the regulators
13
Other regulatorsMyostatin negatively regulates
muscle size
A4 22-43
14
Osteogenesis Bone formation

• Cartilage and bone are derived from several
  sources
• 1) Sclerotome (trunk- ribs, vertebrae) axial
  bones
• 2) Lateral plate mesoderm (limbs)
• 3) Neural crest (head)
• There are two forms of osteogenesis
• 1) Endochondrial ossification (cartilage
  template)
• 2) Intramembraneous ossification (direct
  production of bone)only in the skull

15

• Endochondrial ossification (all other bones)
• Cell types
• 1) Chondrocytes (cartilage formation)
• 2) Osteoblasts (bone formation)
• 3) Osteoclasts (bone resorption)

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Endochondrial ossification
14.13
17
Molecular control of cartilage formation

• 1. Mesenchyme cells ? chondrocytes.
• Sox-9 is required for mesenchyme condensation,
  the first
• requirement for bone formation. The eventual
  size of the
• bone depends on how much mesenchyme condenses

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• 2. Chondrocyte proliferation
• Stimulated by growth factors (IGF) and a hedgehog
  signal

• 3. Chondrocytes ? hypertrophic chondrocytes
• Favored by CbfaI, transcription factor
• Hypertrophy requires estrogens and testosterone
• This sets the stage for bone formation

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Blood vessels bring in chondroclasts (which get
rid of cartilage) and osteoblasts, the bone
precursors
14.13
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(No Transcript)
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Factors required for bone formation
Osteoblasts are bone producing cells they are
brought in via the blood vessels --Require
Cbfa1, a transcription factor, for
differentiation --Without Cbfa1, skeleton remains
cartilagenous
Red bone Blue cartilage
Wild type
Cbfa1 knockout
22
Osteoclasts are derived from haematopoietic stem
cells These cells break down the bone, leaving
an area for bone marrow, which will be site of
blood synthesis (and future synthesis of
osteoclasts)
estrogen testosterone

• Osteoclast numbers are tightly regulated
• Inhibited by estrogen and testosterone