Muscle and Bone formation

Question 1

describe the paradigm of differentation of muscle in 3 stages

Answer 1

1. specification/determination of cells to become myoblasts
2. differentiation of myoblasts into myofibres.
3. maturation of myofibres - acquisition of fibre type specificity

Question 2

1. what is the structure of the Muscle Regulatory Factors? Which domain binds DNA? Which domain enables dimerisation, and what 2 proteins do they form heterodimers with?
2. what is their overall function? How do they perform this function?
3. name the 4 members of the family.

Answer 2

1. basic helix-loop-helix | basic domain binds DNA | helix-loop-helix mediates dimerisation with E12 or E47
2. TFs. They bind to a specific sequence found upstream of genes that encode muscle TFs and contractile machinery proteins.
3. Myf5, MyoD, Myogenin and MRF4

Question 3

1. from which type of mesoderm is skeletal muscle derived from?
2. What part of this mesoderm does skeletal muscle originate from? In which position is this?
3. What is expressed in the mesoderm in Q2?

Answer 3

1. paraxial mesoderm (the somite)
2. dermomyotome (dorsal somite)
3. Pax3

Question 4

1. what signals are responsible for patterning the somite, forming the dermomyotome?
2. From which structures do these signals originate?

Answer 4

1. Shh specifies ventral regions. Low [Shh] specifies the dermomyotomeWnts and BMP4 also specify the dermomyotome
2. Shh from the notochordWnts from the neural tubeBMP4 from the lateral plate mesoderm

Question 5

name the 2 regions of myotome, where they are positioned, what muscles they eventually become, and what signals that induce them.

Answer 5

1. hypoaxial. Lateral myotome. becomes abdominal and limb muscles following ventral migration. induced by wnts and BMP antagonists
2. epaxial. Medial Myotome. becomes the deep back muscles. Induced by wnt signalling and low [Shh]

Question 6

name the order in which MRFs are expressed, and how long they are maintained for

Answer 6

1. Myf5. It is expressed from E8-E12
2. MyoD, which is maintained after birth
3. Myogenin, which too is maintained after birth.

Question 7

1. what is the phenotype of Myf5 KOs?
2. What is the phenotype of MyoD KOs
3. What is the phenotype of MyoD and Myf5 KOs?
4. what is the phenotype of Myogenin KOs?
5. What conclusions can be drawn from these KO experiments?

Answer 7

1. Viable mice with no obvious muscle defects; delay in muscle development until the onset of MyoD expression
2. Viable mice with no obvious muscle defects; increase of myf5 experssion in somites to compensate for the lack of myoD. Deficit in muscle regeneration
3. complete abasence of skeletal muscle; no presence of myoblasts
4. mice die shortly after birth due to a diaphragm deficit. the reduced density of myofibres is replaced by myoblasts
5. Myf5 or MyoD are required to generate myoblasts. Myogenin is required for muscle differentiation.

Question 8

1. describe the relationship between proliferation and differentiation
2. Describe 2 ways that the levels of MyoD and Myf5 are kept relatively low in proliferating cells
3. what is required in order for a cell to differentiate?
4. Describe 2 ways that the process of Q3 is brought about

Answer 8

1. they are mutually exclusive (i.e occur independently of each other)
2. they are phosphorylated by Cdks which makes them more likely to be degradedId interracts with E12/47 to prevent the formation of heterodimers
3. cell cycle arrest
4. Myogenin activates the transcription of p21, proteins which block cell cycle progressionRB must be dephosphorylated for cell cycle arrest. It is usually phosphorylated by Cdks. MyoD interferes with Cdk4 to prevent RB from being phosphorylated

Question 9

1. cells which are destined to become limb muscles must migrate from where to where?
2. What proteins aid this migration?
3. What is the state of specification of cells as they migrate?
4. What occurs once the cells have reached their target? What TFs mediate this? (4)

Answer 9

1. From the dermomyotome to the limb bud
2. Pax3 induces c-met expression. c-met is a receptor for Hepatocyte growth factor that is produced by the limb bud
3. unspecified.
4. organisation into dorsal and ventral muscle masses, and undergo proliferation - Pax3, Meox2, Six1/4

Question 10

1. what are satellite cells?
2. where do they originate from? Where are they found in the developed tissue?
3. what is their role
4. what do they express?

Answer 10

1. adult muscle specific stem cells
2. originate from somites. found in basal lamina surrounding muscle fibres
3. aid the repair of muscle throughout adult life
4. Pax7

Question 11

1. What is the axial skeleton derived from?
2. What is the limb skeleton derived from?
3. what is the cranioskeleton derived from?

Answer 11

1. somites
2. lateral plate mesoderm
3. neural crest cells

Question 12

1. name the 4 distinct regions of the vertebral collumn
2. How do hox genes produce the different identities of these regions?
3. 5’ Hox genes give rise to what structures? 3’ Hox genes give rise to what structures?
4. Which hox genes are expressed first?
5. What is probably responsible for the temporal expression of hox genes?
6. how many cervical vertebrae do all mammals have? How do hox genes regulate the length of mammalian necks?

Answer 12

1. cervical, thoracic, lumbar and saccral
2. the boundaries of these regions corresponds to a boundary of differential Hox gene expression
3. 5’ = anterior; 3’ = posterior
4. 5’
5. linking gene activation to the time that cells spend in the somitic stem cell region
6. 7; Hox genes control the rate of proliferation of hox genes to control the size of the cervical vertebrae.

Question 13

1. what type of ossification is involved in the formation of the axial skeleton? What does this involve?
2. describe the paradigm of differentiation relating to axial skeleton formation
3. from where in the somites is the axial skeleton derived? What is the name of this region?
4. What feedback loop is responsible for dividing the somite into anterior and posterior regions?

Answer 13

1. endochondral ossification. It involves the formation of carteledge which is replaced by bone
2. specification of sclerotomal cellsdetermination of cells forming chondroblastsdifferentiation of chondroblasts forming chondrocytesmaturation of chondrocytes forming hypertrophic chondrocytes
3. anterio-ventral somite. Known as the sclerotome
4. positive and negative loops between delta1 and mesp2. Delta/notch signalling induces mesp2 and delta1. mesp2 inhibits delta1. Eventually through these loops, mesp2 expression is restricted to the anterior half of the somite.

Question 14

1. What TFs are expressed in the sclerotome? Where abouts in the sclerotome are each expressed?
2. Which signals (and from where) induce the expression of these TFs?
3. Name the 4 different parts of the axial skeleton and from which part of the sclerotome/somite each is derived.

Answer 14

1. Pax1 and Pax9. Pax1 is expressed medially, and Pax9 laterally
2. High [Shh] as experienced by the ventral somite induces Pax1 and Pax 9. BMP4 from the lateral mesoderm restricts Pax1 expression to the medial sclerotome, thus allowing Pax9 to be expressed in lateral regions
3. neural arches (dorsal sclerotome)distal ribs (dermomyotome)vertebral bodies (medial sclerotome)proximal ribs (lateral sclerotome)

Question 15

1. What is the phenotype of the Pax1 KO?
2. What is the phenotype of the Pax9 KO?
3. What is the phenotype of the Pax 1 & Pax9 KO?

Answer 15

1. viable mouse; some abnormalities in the vertebral collumn, scapula and sternum
2. mice die shortly after birth; abnormalities in craniofacial and limb skeleton as Pax9 is expressed in these precursors too
3. complete lack of derivatives from ventral sclerotome - vertebral bodies, intravertebral discs, proximal ribs lacking.

Question 16

1. What do sclerotomal cells do as they become determined into chondroblasts?
2. What signal induces the proliferation of chondrocytes?
3. What signal is required for the production of cartilage matrix proteins by chondrocytes?

Answer 16

1. migrate to and condense around the neural tube
2. BMPs
3. Sox9

Question 17

1. Describe the stages of endochondral ossification
2. Name 3 cartilage producing signals and their roles
3. Name 2 bone producing signals and their roles
4. What signalling pathway maintains the balance between bone and cartilage producing signals and how?

Answer 17

1. 1) chondrocytes undergo terminal differentiation and become hypertrophic
   2) hypertrophic chondrocytes undergo apoptosis. Apoptosing cells release chemicals which attract blood vessels into the area
   3) the infilitrating blood vessels bring osteoblasts with them, which together form the bone marrow
   4) osteoblasts replace the dissapearing cartilage, forming the primary ossification centre at the diaphysis
   5) blood vessels enter the epiphyses as chondroblasts undergo apoptosis here. The secondary ossification centres are formed here, leaving a cartilage growth plate
2. Sox6 is involved in condensation of chondroblasts. Sox6, L-Sox5 and Sox9 are involved in differentiation. Sox 9 inhibits the maturation of chondrocytes
3. Runx2 is involved in maturation of chondrocytes
4. Runx2 and Osterix are involved in determination and differentiation of osteocytes
5. Balance is maintained by Wnts: promotes bone formation and inhibits cartilage formation.

Question 18

1. Name the 6 long bones
2. Where are growth plates located. What do they act as?
3. How are cells of the growth plates organised?
4. Which 3 signals maintain the pool of proliferating chondroblasts?
5. How do these signals do so?

Answer 18

1. Humerus, Radus, Ulna, Femur, Tibia and Fibula
2. epiphyses of long bones. They act as a reserve pool of cartilage cells for growth
3. stratified organisation of chondroblasts, chondrocytes and hypertrophic chondrocytes
4. IHh, PTHrP and FGFR3
5. Cells which progress towards terminal differentiation produce IHh. IHh signals for cells to produce PTHrP. The PTHrP receptor is only found on chondroblasts

PTHrP stimulates chondroblast proliferation and prevents differentiation

FGFR3 limits proliferation (thus growth) by inhibiting IHh.