14.10

Somite maturation over time: the somite separates into four regions

Class 16: Differentiation of Somites: Muscle and Bone

G 14.10

dermatome Hypaxial myotome

sclerotomeMigrating myoblast

Epaxialmyotome

Cell types within the somite

Multiple signals determine derivatives of the somite Epaxial myotome: makes back muscles

Reguires Wnts from neural tubeShh from notochord

Hypaxialbody wall, limb muscles

Hypaxial myotome: makes limb and body wall musclesRequires BMP4, FGF from lateral plate mesoderm

Wnts from overlying ectoderm

14.11

Sclerotome: makes bone (vertebrae and ribs)Requires Shh from notochord

How is muscle made?The discovery of a “master” regulatory factor for muscle

MyoD expression in the E11.5 mouse embryo

somites

forelimb

hindlimb

eye

bHLH transc. FactorsShow binding domain, etc

A “master regulatory gene” for muscle differentiation:MyoD, bHLH family of transcription factors

14.12

Myogenesis: Stages in determination and differentiation of myoblasts

Myotome

(only proliferating)

Myoblast

(starting to differentiate)

FGF

no FGF

Three additional members of the MyoD family were found,each with specific roles and different expression patterns

Myf-5: expressed in epaxial (back) myotome

MRF-4: expresed as myoblasts differentiate intomyotubes and myofibers

Myogenin: expresed as myoblasts differentiate intomyotubes and myofibers

MyoD: expressed in hypaxial (limbs) myotome

Phenotypes of mouse knockout mutants for myogenic genes

MyoD -/- Normal muscle (developmental delays)

Myf5 -/- Normal muscle (developmental delays)

MyoD -/- ; Myf5 -/- Paralyzed, no myoblasts or muscleMyogenin -/- Myoblasts, but few myotubes

MRF4 -/- Muscle fibers (but delayed)

14.12

Myogenesis: Stages in determination and differentiation of myoblasts

Committed, express MyoD but still dividingMrf-4

Myogenin

MyoD family members are only active as heterodimerswith other bHLH proteins

Regulation of MyoD family members

Basic Helix 1 Loop Helix 2

MyoD

E12

DNACONTACT

DIMERIZATION

CANNTGGTNNAC

MyoD E12

Timing of differentiation is controlled by levels of the regulators

NegativeIdMist1MyoR

(don’t bind DNA)

PositiveE proteins (E12 and E47)(bind DNA)

There are both positive and negative regulators of MyoD family members

A4 22-43

Other regulators:Myostatin negatively regulates muscle size

Belgian Bluemyostatin mutants

An interesting tidbit:

Muscle can regenerate because of a unique population of cells that normally are “quiescent”

Satellite Cells

Inactive stem cells that are triggered by growth factors to undergo division and produce myoblaststhat then develop into myofibers

Studied here in MCDB by Dr. Brad Olwin

A regulatory pathway for myogenesis

Myotome

Myoblasts

Myotube Striated myofiber

Wnt

Shh

Pax3

MyoD

Myf5

Myogenin

MRF4

Myosin, actin, etc.

Somite

FGF

Myofiber

dermatome Hypaxial myotome

sclerotomeMigrating myoblast

Epaxialmyotome

Osteogenesis: Bone formation

Cartilage and bone are derived from several sources:

1) Sclerotome (trunk- ribs, vertebrae)

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)

Axial bone derives from the sclerotome

14.11

Intramembranous ossification: skull

Endochondrial ossification (all other bones)

Cell types:

1) Chondrocytes (cartilage formation)

2) Osteoblasts (bone formation)

3) Osteoclasts (bone resorption)

14.13

Endochondrial ossification

Molecular control of cartilage formation

1. Mesenchyme cells à chondrocytes.

Sox-9 is required for mesenchyme condensation, the firstrequirement for bone formation. The eventual size of the bone depends on how much mesenchyme condenses

2. Chondrocyte proliferation

•Stimulated by growth factors (IGF) and a hedgehog signal

3. Chondrocytes à hypertrophic chondrocytes

•Favored by CbfaI, transcription factor

•Hypertrophy requires estrogens and testosterone

•This sets the stage for bone formation

14.13

Blood vessels bring in chondroclasts (which get rid ofcartilage) and osteoblasts, the bone precursors

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: boneBlue: cartilage

Wild type Cbfa1 knockout

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)

Osteoclast numbers are tightly regulated

•Inhibited by estrogen and testosterone

estrogentestosterone

14.13

Blood vessels bring in the osteoclasts, which “remodel” the bone