Apoptosis

1
Apoptosis
fig.cox.miami.edu
2
Background

• Cell suicide
• Construction,
  maintenance,
  repair
• All nucleated
  cells

ghr.nlm.nih.gov
3

• Vogt 1842
• J.F. Kerr et al (1972)
• Genetically controlled
• Molecular activators
• Evolutionarily conserved

C. Elegans expasy.org
4

• 4 functional group
  genes
• Ced-3 ? caspases
• Ced-4 ? Apaf-1
• Ced-9 ? Bcl-2
• Egl-1 ? BH3

5

• Human viral, degenerative diseases
• Therapy - cancer

2006 Wikipedia CD Selection
6
Embryonic development

• Physiologically, genetically controlled
• Malformations
• Different stages, organs
• Sculpts organs
  morphogenetic
• Removal of cells -
  histogenetic

www.ccs.k12.in.us
7
Appendages

• Mesoderm
• Amount of cells for
  skeleton
• Between digits
• Chondrogenetic skeletal
  condensations

8

• Ectoderm
• Apical ectodermal
  ridge (AER)
• Mesenchymal cells

www.tmd.ac.jp
9
Fish

• Many organs
• Sensory organs, brain morphogenetic
• Fins epidermal cells, cartilage
• Median fin fold unpaired dorsal, anal, caudal

10

• 20 hrs post fertilization (hpf)
  median fin fold appears
• 22 hpf distal parts of fin
  fold, proximally
• 24 hpf distal tip
• Present until 72 hpf
• Not morphogenetic

Hpf Median fin fold
12 14 16 - - -
20 22 24 0 11.0 /- 5.57 7.00 /- 6.08
30 36 48 13.67 /- 13.50 20.00 /- 6.24 12.00 /- 6.00
60 72 20.33 /- 8.08 5.00 /- 2.83
Apoptosis in median fin fold Cole and Ross, 2001
11
Amphibians

• Not with free digits
• Some anurans, urodeles
  absent
• Some salamanders
  similar to amniotes
• X. laevis different proliferation rates in
  digital, interdigital
• Hindlimb comparable

12
Reptiles

• Patterns correlate with adult
  limb morphology
• Turtles distal interdigital
  areas
• Lizards interdigital
• Apoptosis digit formation
  1st in amniotes (Fallon and
  Cameron, 1977)

13

• Ectoderm of AER
• Cells undifferentiated, proliferating
• Snakes massive apoptosis

14

• Chameleon
• Autopodial cleft
• Specialized
  interdigital cell death
• Begins early, wide
  along distal margin

15
Avians

• Skeletal
  primordia of
  limbs
• 2 areas undifferentiated mesenchyme
• Anterior, posterior margins proximal segment of
  limb
• Anterior, posterior necrotic zones (ANZ, PNZ)
• Reduction in digit number

www.dls.ym.edu.tw
16

• Opaque patch (OP) central mesenchyme
• 2 pieces of zeugopod
• Digit formation -
  mesenchyme
  between rays
• Interdigital necrotic
  zone (INZ)

www.dls.ym.edu.tw
17

• Constant w/in species
• Different for different
  species
• Correlate w/ limb
  morphology
• Free digits throughout interdigital space
• Webbed feet distal interdigital
• Free digits
  membranous fold
  central interdigital

18

• Inhibited syndactyly
• AER spatial, temporal extension of limb

Kingfisher www.turtletrack.org
19
Mammals

• Ectodermal
  morphogenetic
• AER postaxial, preaxial
  margins
• Regression of extreme ends
• Inhibition - polydactyly

www.nature.com
20

• Postaxial ridge digit V
• Interdigital digit IV
• Later, entire length
• Decreases except digit I

www.gsc.riken.go.jp
21

• Footplate foyer preaxial primaire (fpp)
• Similar to ANZ
• Reduces quantity of
  preaxial mesodermal cells
• Talpa (mole) fpp absent,
  falciform digit

fpp
www.nature.com
www.palaeos.com
22

• Subridge mesoderm foyer marginal I (fmI)
  preaxial margin
• Digits 1-3 in forelimb
• 1-1/2 digit 2 in hindlimb
• Foyer marginal V (fmV) postaxial
  margin
• Digit 5 to border of 4
• Growth of digital buds
• Decrease in influence of ectodermal layer

fmI
fmV
23

• Interdigital mesoderm
  separation of digits
• 2 waves superficial layer of subectodermal
  cells
• Between precartilaginous
  rudiments of phalanges

24
Mechanisms

• Bone morphogenic proteins (Bmps)
• Transforming growth factor ß superfamily
• Bmp-2, 4, 7, 5 undifferentiated limb mesoderm,
  interdigital mesoderm, AER
• Coincide with cell death

25

• High redundancy
• Regulated by Bmp
  antagonists
• Noggin, gremlin,
  DAN, Drm
• Gremlin ducks, down regulated in chicks prior
  to INZ

26

• Bmps limb patterning,
  regulate chondrogenic
  differentiation
• Signal
  serine/threonine
  receptor kinase with
  type I and II receptors
• Binding association of 2 receptors
• Phosphorylation of type I by II
• Propagation of intracellular signal

www.medscape.com
27

• Chondrogenic effects
  type Ib receptor
• Type Ia control of
  apoptosis
• Interdigital induction
  of Ib ectopic digit

arthritis-research.com
28

• Bmps signal through
  Smads
• Bmp binds to receptor
• Smad cascade
  BMP-responding
  smads 1, 5, 8
• Co-smad 4
• Inhibitory smads 6, 7
• Translocated to nucleus
• Activate transcription

29

• Also signal through
  MapK pathway
• Erk, Jnk, p38 kinase
  mediate Bmp
  signaling

MapK pathways e-kisstoth.staff.shef.ac.uk
30

• Limb
  caspase-3, 9, 2
• Death
  Inducer-Obliterator-1
  (DIO-1)
• Growth Arrest
  Specific1, 2 (Gas1, 2)
• Apaf-1

arthritis-research.com
31

• Bax Bcl-2 family
  proapoptotic
• Antiapoptotic Bcl-2,
  Bcl-x, A1 digital rays
  not interdigits
• Before apoptosis Bag-1 expresses antiapoptotic
  protein
• Binds to Bcl-2 in interdigits
• Defender Against apoptotic cell Death (Dad-1)
• Syndactyly

32

• Fgf signaling
  outgrowth of limbs
• Cooperate with
  Bmps
• Blocked Bmps do
  not trigger
  apoptosis
• Webbed feet of
  ducks decrease
  in Fgf
• Fgfs activate ERK

www.nature.com
33

• Retinoic acid signaling
  limb patterning
• Acts with Bmps
  interdigital regions
• Promotes apoptotic
  effects of Bmps
• Inhibits chondrogenic
  effects
• Bmps induce
  apoptosis, promote
  cartilage growth

a-b dying cells c-d macrophage distribution e-f
macrophage specific antibodies g-h S phase
nucei Dupe et al, 1999
34
Joint Formation
35

• Internal skeleton support, locomotion
• Joints classified by structure, degree of
  movement
• 1. synarthrosis joined by cartilage
• 2. schizarthrosis interzone contains single
  (small ) of cavities
• 3. hemiarthrosis single joint cavity, elements
  united around perifery
• 4. eudiarthrosis separate
  articulating elements, cavity limited by
  synovial tissue

36

• Degree of movement a joint
  allows
• 1. Synarthrosis
  no movement
• 2. Amphiarthrosis limited
  movement
• 3. Diarthrosis freely movable

37

• Diarthroidal joints aquatic to terrestrial life
• Agnatha to
  Gnathostomata
  hinged mandible
• Greater range of prey

38

• Agnathans branchial
  arches
• Mandibular arch
  chondrocranium
  jaws
• Upper palatoquadrate
  lower mandibular cartilage
• Mammals - malleus incus
  - diartroidial

39

• Chondrichthyes
• Charchariniformes,
  Squaliformes
  hemiarthrosis
• Holocephali more
  diarthroidial
• Synovial membrane on
  one side
• More analysis

40

• Rajidae diarthroidial
• Arose in cartilaginous fish
• May be lost in
  elasmobranches

41

• Osteichthyes some
  may have lost diarthroses
• Quadrate/mandibular -
  microscopic structure
• Polypterus, Protopterus

Haines, 1937
42

• Lepidosteus (longnose
  gar)
• Layer of calcified cartilage
• Hypertrophic chondrocytes
  integrated into bone
• Hyaline cartilage
• Articular fibrocartilage birds, mammals
• No fibrous capsule loose
  connective tissue
• Synovial membrane
  bilayered

AF
CC
HC
Haines, 1942
43

• Early dipnoans bony, supported by overlying
  cartilage
• Living secondary modification
• Fins - no diarthrosis
• Synarthrosis distal, smaller
  joints
• Schizoarthrosis, hemiarthrosis
  proximal, larger joints

44

• Amia calva (bowfin)
  proximal radial/girdle
  diarthroidal?
• Joint cavity, minimal
  connective tissue,
  2-layered synovium
• Modern bony fish more diarthroses
• Maneuverability swimming, feeding
• Larger size large joints at base of fins

45

• Tetrapod limbs
  diarthroidial
• Urodeles, anura
  distal joints
  synarthroses
• May be secondary modification

46

• Primitive structure
  no joint capsule
  supporting 2-layered
  synovial membrane
• Amphibia, reptilia
• Fibrous/fibrocartilaginous
  articulating surface overlying hyaline cartilage
  of epiphysis - birds

Crocodile knee Haines, 1942
47

• Crocodilus, Sphenodon,
  lizards primitive
• Cruciate ligaments,
  menisci, single joint
  cavity femur/tibia/fibula
• Chelonians firm
  articulation between
  median condyle of
  femur/tibia
• Reduction in medial meniscus

48

• Urodeles reduced/lost cavity, menisci,
  ligaments
• Marsupials,monotremes
  femora-fibular
  articulation
• Joint cavity subdivided
  by connective tissue
• Eutherians articulation
  lost
• Femur closely bound to
  tibia

49

• Histological features of joint epiphyses
• Bony fish
  cartilaginous
  epiphyses at end
  of diaphyses
• Articular surfaces fibrous
• Perichondrium?
• Mass of rounded chondrocytes surrounded by
  cartilage matrix
• Metaphyses become flattened, hypertrophied

50

• Matrix may become
  calcified
• Reabsorbed by elements
  of bone marrow marrow
  processes
• True endochondral
  ossification

51

• Calcified cartilage in
  center of epiphyses of
  epibranchial bone
• Forerunner of secondary
  center of ossification
• Closing plate of
  endochondral bone
• Bony fish true epiphysis endochondral growth
  mechanism

52

• Early tetrapods
  cartilaginous epiphyses
• Lacked secondary centers
  of ossification
• Chelonia, Crocodilia retain
  primitive condition
• Modifications for land dwelling
• Reduction in zone of round cells
• Flattened zone closer to articular surface
• Firmer epiphysis

53

• Tuatara most primitive
  secondary ossification
• Large masses of calcified
  cartilage
• Greater part of adult
  epiphysis
• Thin layer of articular cartilage
• Flattened cell zone partitioned into columns
• Division of founder population at top of column
• Progeny lie beneath mother cell

54

• Mammals alignment
  occurs early
• Reptiles/birds initially
  not aligned
• Loose alignment in
  postembryonic development
• Maintained during hypertrophy, calcification
• Only septa between hypertrophied cells calcifies
• Form templates for endochondral bone

55

• Noncalcified septa
  broken down by
  metalloproteinases
• Hypertrophs undergo
  apoptosis,
  transdifferentiation into
  ostoblasts

56

• Cavity greater range of
  motion
• Schizarthrosis primitive
  condition?
• Mechanism differential
  hyaluronan (HA) synthesis
• Mechanical stimuli
• Glycosaminoglycan HA, CD44 differentially
  expressed at joint interzone, articular surfaces

57

• Diphospho-glucose dehydrogenase (UDPGD) increases
  prior to cavitation in interzone
• Articular surfaces, synovium
  during cavitation
• UDPGD UDP-glucuronate HA
• HA synthesis increases at time of separation
• HA CD44 adhesion, separation
• Depends on concentration
• CD44 - interzone, articular surfaces increased
  HA synthesis - separation

58

• Mechanical strain influences HA synthesis
• More strain increases HA, UDPGD, CD44
• HA displaced from receptor fused joints

59

• Evolution of higher vertebrates
• Ability to respond to mechanical
  cues of joint motion
• Increase in HA synthesis
• Accumulation between
  opposing elements

60

• Terrestrial evolution reduction in number of
  joints
• Secondarily aquatic hyperphalangy
• Increased number of joints
• Rare in terrestrial amniotes

61

• Early ichthyosaurs 2-4-4-4-1
• Later up to 30
• Early cetaceans little hyperphalangy
• Extant up to 14
• Better maneuverability, navigation

62

• Digit length, phalange/joint
  number AER, Fgfs
• Chick Fgf8 in AER first
  switched off over digit IV,
  then II, then III
• Correlates with phalange
  number
• White sided dolphin
  maintained over digits II
  and III

63

• Fgf8 regulated by Shh in
  ZPA
• Ihh condensing cartilage
  of digits
• Signals for joint position mesenchyme posterior
  to each digit

64
Scenarios in joint formation

• Long bone elements
• Cartilage differentiates
  across joint locations
• Chondrocytes flatten
• Matrix becomes nonchondrogenic type I, III
  collagens, little proteoglycan
• Interzone signaling center, acts on opposing
  elements

65

• Tarsals, carpals
• Chondrogenesis center of condensations
• Expand through matrix
  accumulation
• Periphery cells stretch
  form boundary perichondrium
• Abut perichondrium of neighboring element
• Interzone present, not clearly defined

66

• Secondary cartilaginous
  joints
• Bone formation before
  cartilage
• Mechanical stimulation
  progenitor cells in periosteum become
  chondrogenic
• Form joint with neighboring cartilage element
• GDF-5 - primary joint formation

67

• Molecular mechanisms long
  bones
• Reversal of chondrogenic
  phenotype
• Blocked prochondrogenic
  signaling
• Noggin, GDF-5, Chordin
  inhibit Bmps in interzone

68

• Bmp-7
  prochondrogenic
• Perichondria of
  cartilaginous primordia
• Absent at presumptive
  joint
• Bmp-2, Bmp-4 similar

69

• GDF-5 2 roles in
  skeletogenesis
• Promotes condensation
  of mesenchyme
• Promotes proliferation of
  chondrocytes in epiphysis
• Maintenance, early
  development of some
  joints
• Mutant mice joint
  missing

70

• GDF-6 carpals, tarsals
• Knockouts no wrist joints
• GDF-5 marks digit joints
• GDF-5/6 knockouts form
  joints, lost secondarily
• Maintenance of joints

71

• Contact - fish homologue of GDF-5
• Between dorsal fin and fin radials
• Evolution of joint morphogenesis

72

• Wnt9a interzone
• Upstream of GDF-5, CD44,
  chordin, autotaxin
• Wnt9 hagfish, thresher
  shark
• Cux-1 inhibits
  chondrogenesis
• Interzone

73

• Joint number maintenance
  of joint specifying signals
• Increased Fgf signaling
  loss of joints
• Hyperphalangy duplication
  of Wnt9a
• More distal expression due
  to prolonged survival of AER
• Extended expression of Fgf-8

74
Fin dermoskeleton

• Unmineralized actinotrichia
  segmented, bony lepidotrichia
• Joined by collagenous
  ligaments
• Evx-1 related to pair-rule
• Precedes joint formation
• Marks developing joints
• May be involved in joint specification