somite maturation over time: the somite separates …3) neural crest (head) there are two forms of...
TRANSCRIPT
![Page 1: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/1.jpg)
14.10
Somite maturation over time: the somite separates into four regions
Class 16: Differentiation of Somites: Muscle and Bone
![Page 2: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/2.jpg)
G 14.10
![Page 3: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/3.jpg)
dermatome Hypaxial myotome
sclerotomeMigrating myoblast
Epaxialmyotome
Cell types within the somite
![Page 4: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/4.jpg)
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
![Page 5: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/5.jpg)
How is muscle made?The discovery of a “master” regulatory factor for muscle
![Page 6: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/6.jpg)
MyoD expression in the E11.5 mouse embryo
somites
forelimb
hindlimb
eye
![Page 7: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/7.jpg)
bHLH transc. FactorsShow binding domain, etc
A “master regulatory gene” for muscle differentiation:MyoD, bHLH family of transcription factors
![Page 8: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/8.jpg)
14.12
Myogenesis: Stages in determination and differentiation of myoblasts
![Page 9: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/9.jpg)
Myotome
(only proliferating)
Myoblast
(starting to differentiate)
FGF
no FGF
![Page 10: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/10.jpg)
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
![Page 11: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/11.jpg)
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)
![Page 12: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/12.jpg)
14.12
Myogenesis: Stages in determination and differentiation of myoblasts
Committed, express MyoD but still dividingMrf-4
Myogenin
![Page 13: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/13.jpg)
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
![Page 14: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/14.jpg)
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
![Page 15: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/15.jpg)
A4 22-43
Other regulators:Myostatin negatively regulates muscle size
Belgian Bluemyostatin mutants
![Page 16: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/16.jpg)
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
![Page 17: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/17.jpg)
A regulatory pathway for myogenesis
Myotome
Myoblasts
Myotube Striated myofiber
Wnt
Shh
Pax3
MyoD
Myf5
Myogenin
MRF4
Myosin, actin, etc.
Somite
FGF
Myofiber
![Page 18: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/18.jpg)
dermatome Hypaxial myotome
sclerotomeMigrating myoblast
Epaxialmyotome
Osteogenesis: Bone formation
![Page 19: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/19.jpg)
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
![Page 20: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/20.jpg)
14.11
Intramembranous ossification: skull
![Page 21: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/21.jpg)
Endochondrial ossification (all other bones)
Cell types:
1) Chondrocytes (cartilage formation)
2) Osteoblasts (bone formation)
3) Osteoclasts (bone resorption)
![Page 22: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/22.jpg)
14.13
Endochondrial ossification
![Page 23: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/23.jpg)
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
![Page 24: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/24.jpg)
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
![Page 25: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/25.jpg)
14.13
Blood vessels bring in chondroclasts (which get rid ofcartilage) and osteoblasts, the bone precursors
![Page 26: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/26.jpg)
![Page 27: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/27.jpg)
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
![Page 28: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/28.jpg)
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
![Page 29: Somite maturation over time: the somite separates …3) Neural crest (head) There are two forms of osteogenesis: 1) Endochondrial ossification (cartilage template) 2) Intramembraneous](https://reader033.vdocument.in/reader033/viewer/2022042803/5f42b2eaebd4736f4833c7c0/html5/thumbnails/29.jpg)
14.13
Blood vessels bring in the osteoclasts, which “remodel” the bone