Limb development

Question 1

Limb - model system for human patterning formation

Answer 1

Vertebrate limb = intricately patterned 
Obvious pattern/axes 
Dispensable 
Defects - easily recognisable 
(many rare genetic disorders affect limb patterning)

Question 2

What is patterning?

Answer 2

Creation of specific structures according to a pattern (in specific locations)

Question 3

Axes

Answer 3

Proximodistal: shoulder to fingertip
Dorsoventral: back to palm of hand
Anteroposterior - thumb to little finger

Question 4

Proximodistal patterning

Answer 4

Development of limb bud
5 week period, week 4-8
Upper limb develop 1-2 days ahead of lower
End limb development - upper and lower almost synchronised

Question 5

Initiation of limb bud development (P-D)

Answer 5

Cont. proliferation of parietal lateral plate mesoderm, stimulate by FGF 10
Upper limb bud: appear in lower cervical region at 24 days
Lower limb bud: appear in lower lumbar region at 28 days

Question 6

Structure of limb bud

Answer 6

Outer ectodermal (epithelial) cap 
Inner mesenchymal core - derive from parietal lateral plate mesoderm (bones + CT of limbs)

Question 7

Apical ectodermal ridge (P-D patterning)

Answer 7

Ectoderm along distal border of limb bud thickens = AER at dorsal-ventral boundary of limb bud
Induced by underlying somatic mesoderm
Essential for outgrowth of limb

Question 8

FGF feedback loop

Answer 8

FGF10 express in lateral plate mesoderm, induce AER, release FGF4 + 8, maintain FGF10 = continue outgrowth of limb bud (maintain progress zone)

Question 9

Undifferentiated/progress zone

Answer 9

AER exert inductive influence on adjacent mesenchyme - cause remain popn. undifferentiated rapidly proliferating cells

Question 10

Proximodistal development of limb

Answer 10

Limb grows, cells farther from AER start differentiating (cartilage + muscle)
Develop proximodistally to 3 components:
Stylopod, zeugopod, autopod

Question 11

Stylopod

Answer 11

Humerus and femur (most proximal)

Question 12

Zeugopod

Answer 12

Radius, ulna + tibia, fibula

Question 13

Autopod

Answer 13

Carpals, metacarpals, digits/ tarsals, digits/ metatarsals (most distal)

Question 14

Week 6

Answer 14

Mesenchymal core form 1st hyaline cartilage models
Terminal portion of limb bud flattens = hand plates/ foot plates (separate from proximal segment by circular constriction)
Second constriction - divide proximal portion into 2 segments
Cont. outgrowth influence by 5 segments of AER
Condensation of mesenchyme = cartilaginous digital rays
Death tissue between rays
Complete set of cartilaginous models (end week 6)

Question 15

Digit formation

Answer 15

Cell death separates AER into 5

Question 16

Limb rotation: Week 7

Answer 16

Upper - 90 degree laterally
Ext. muscles on lateral, posterior surface, thumbs lateral
Lower - rotate 90 degree medially
Ext muscles on anterior surface, big toe medial
Produce spiralling dermatomes

Question 17

AER experiments (chicks): P-D patterning

Answer 17

AER = diff. programming abilities depending on stage limb development
(1) Remove AER:
Early - Stylopod only
Mid - S + Z
Late S + Z + A
(2) Replace AER w/ bead of FGF = restore normal limb
(3) Remove AER + progress zone from early limb development (S + Z) and transplant into late = S + Z + S + Z + A
(4) Remove AER + PZ from late and transplant to early = truncated limb (e.g. S + Z + hand)
FGF = signal and patterning

Question 18

Thalidomide

Answer 18

May affect cells in progress zone, not know how long in contact w/ AER
As limb grows out, cells drop out of PZ and differentiate - thalidomide may disrupt
Result = phocomelia (absence/ deformity long bones, intestines + cardiac anomalies)

Question 19

Patterning signalling centres

Answer 19

Patterning needs 1+ spatially localised signalling centres
Multiple centres specify position
Limited range signal molecules used and reuse in developmental patterning
Cells respond in cell-type specific (CTS) way to signals, active CTS transcription program
Cells already partially specialised - allow limit range of signals for wide range of cell types
Develop higher eukaryotes need establish many signal centres

Question 20

HOX genes

Answer 20

Transcription factors
High functional conservation of homologues (same relative positioning) in craniocaudal axis
Express at different points along body axis
Multiple copies in humans - reduce affect of mutations, redundancy
As limb bud grows, functional domains progressively restricted:
HOX9/10 express in stylopod
HOX11 = Zeugopod
HOX13 = autopod

Question 21

HOX11 knock in mouse

Answer 21

Show broad expression throughout distal limb bud mesenchyme early in development
Expression rapidly restricted to zeugopod

Question 22

Regenerate tail stump of balloon frog

Answer 22

Treat w/ retinoic acid
Induce expression of HOX genes
Regenerate legs

Question 23

Control of anteroposterior patterning

Answer 23

Zone of polarising activity (ZPA): cluster mesenchymal cells at posterior border of limb
Secrete Shh signal (originally thought retinoic acid)

Question 24

Sonic hedgehog (Shh)

Answer 24

Contribute to specification of anteroposterior axis
May induce TBF-beta signals
Morphogen = diff. affect on cell fate at diff concentrations

Question 25

ZPA mirroring (experimental)

Answer 25

ZPA on posterior side Transplant another ZPA onto anterior = mirror image duplication (creates opposing Shh concentration gradient to original)

Question 26

How do we know Shh is the signal from ZPA? (experimental)

Answer 26

Put Shh gene into cells, replace ZPA w/ cells, see if mirror image duplication occur Experiment w/ concentrations by changing no. cells

Question 27

Dorsoventral patterning (experimental)

Answer 27

Dorsal ectoderm = signalling centre | Chicks: remove, rotate, replace dorsal ectoderm = invert axis

Question 28

Wnt7a knock out mouse (dorsoventral patterning experimental)

Answer 28

Ventral structures on both sides (pads) | Wnt7a - induce activity of Lmx1 (TF)

Question 29

Lmx 1 mutation

Answer 29

Nail-patella syndrome: No patella Nails not fully formed

Question 30

Tbx transcription factors (experimental)

Answer 30

Tbx5 - specification of forelimb Tbx4 - specification of hindlimb Express both in same limb = 1 limb bud forms fore and hind limb Put Tbx4 into forelimb of chick = become hindlimb Mutate Tbx 3 and 5 = meromelia (partial limb loss)

Question 31

Bone development

Answer 31

Mesenchyme in bud condenses, cell differentiate to chondrocytes Week 6 = first hyaline cartilage models from by chondrocytes Endochondral ossification begin at end embryonic period: start at primary ossification centres diaphysis (long bone), progress to end of cartilaginous model At birth: diaphysis = completely ossified, epiphyses (ends) = cartilaginous Secondary ossification centres form in epiphyses, temporary cartilaginous plate between diaphyseal and epiphyseal ossification centres = epiphyseal plate - allow bones to grow after birth Endochondral ossification cont. both sides of plate Bone reach full length - epiphyseal plate disappear, epiphyses join w. shaft

Question 32

Development of different types of bone

Answer 32

``` Long bone: epiphyseal plate at each extremity Smaller bone (e.g. phalanges): plate at one extremity Irregular bone (e.g. vertebrae): several primary and secondary ossification centres ```

Question 33

Hypo-secretion of growth hormone

Answer 33

Pituitary dwarfism

Question 34

Endocrine control of postnatal longitudinal bone growth

Answer 34

Growth hormone - secrete by anterior pituitary gland

Question 35

Hyper-secretion of growth hormone

Answer 35

Giantism - XS longitudinal bone growth, originate in childhood before epiphyseal plates ossify

Question 36

Limb musculature

Answer 36

Derive from dorsolateral cells of somites (myotome), migrate into limb to form muscle Muscle components initially segmented according to somite cells derive from Elongate limb bud: muscle tissue split to flexor (ventral) and extensor (dorsal) components + splittings/ fusions = 1 muscle form from +1 original segment Complex pattern of muscle determine by CT derived from lateral plate mesoderm

Question 37

Segmental positioning of limbs

Answer 37

Upper limb bud: opp. lower 5 cervical + upper 2 thoracic segments Lower limb bud: opp. lower 4 lumbar + upper 2 sacral segments

Question 38

Innervation of limbs

Answer 38

``` Buds form, ventral primary rami (branch from spec. spinal nerve) penetrates mesenchyme Form plexus (brachial = upper, lumbosacral = lower) Then form dorsal/ventral branches derived from spec. spinal segment Branches in retrospective divisions unite = large dorsal/ ventral nerves: radial nerve = combine dorsal segmental branches (supply extensor), ulnar, median nerves = combine ventral branches (supply flexor muscles) Nerve enter limb bud and immediately establish contact w/ differentiated mesodermal condensation: need early contact between nerve and muscle cells for functional differentiation, innervate nearest unoccupied territory of muscle, rostral nerves innervate proximal muscle first, subsequent nerves move distally along limb to innervate unoccupied muscle ```

Question 39

Spinal nerves

Answer 39

Important for differentiation/ motor innervation of limb muscles AND sensory innervation of dermatomes Original dermatomal pattern change growth/rotation of extremities

Question 40

Limb abnormalities

Answer 40

Genetic + environmental factors Reduction defects Duplication defects Malformation/ dysplasia

Question 41

Reduction defects (limb abnormalities)

Answer 41

``` Amelia = complete absence 1+ extremity Meromelia = parts missing Ectrodactyly = absence digit ```

Question 42

Duplication defects (limb abnormalities)

Answer 42

Polydactyly = + 5 fingers (often lack proper muscle connections)

Question 43

Malformation/ dysplasia (limb abnormalities)

Answer 43

Syndactyly = malformed digits (fail remove mesenchyme between digits)

Question 44

Cleft hand and foot (clinical)

Answer 44

Patterning defect Rare genetic cause (e.g. Wnt10B) Abnormal cleft between 2nd and 4th metacarpal bones 3rd metacarpal + phalangeal bones almost always absent Thumb/index + 4th/5th fingers = fused