Updated: 5/2/2019


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Limb Development
  • Overview
    • the appendicular system forms between the 4-8 weeks of gestation
    • limb bud development 
      • appears to be under the control of fibroblast growth factors (FGF)
      • enlargement of the limb bud is due to the interaction between the apical ectodermal ridge (AER) and the mesodermal cells in the progress zone.  
      • first identifiable by transvaginal ultrasound at 8 weeks 
  • Steps of limb development  
    • notochord expresses Shh
    • Shh regulates limb bud formation
      • limb bud is combination of lateral plate mesoderm and somatic mesoderm
      • growing outwards into ectoderm (called apical ectodermal ridge)  
      • limb bud formed at embryonic stage 12 (26 days after fertilization)
    • mesenchyme condenses into preskeletal blastemal at core of limb bud
    • chondrification occurs where mesenchyme differentiates into chondrocytes
      • All upper limb bones are endochondral except distal parts of distal phalanges (membranous)
      • From proximal (humerus, 36 days after fertilization) to distal (distal phalanges, 50 days)
      • Factors required for chondrification
        • transcription factors – Sox-5, Sox-6, Sox-9 
        • transforming growth factor superfamily – TGF-b, BMP-2
        • FGF family
          • receptor mutation leads to acrocephalosyndactyly (Apert syndrome)
          • patients with severe craniofacial features have mild hand syndactyly (gain of function in FGFR2c affinity for FGF2 expressed in craniofacial area )
          • patients with mild craniofacial features have severe hand syndactyly (loss of function in FGFR2c specificity for FGF2, and is now able to bind FGF10, more expressed in hands)
          • retinoids
          • hedgehog gene products
          • PTHrP
          • cadherins
          • WNT5a and WNT7a
    • Formation of joints requires repression of chondrogenesis at sites of future joints
      • proteins involved – WNT4, WNT14, growth and differentiation factor 5 (also known as cartilage-derived morphogenetic protein 1)
      • shoulder interzone appears at 36 days, hand interzones appear at 47 days
    • Finger separation
      • digital rays are evident within hand paddle at stage 17 (41 days)
      • interdigital mesenchyme cells undergo programmed cell death (stage 19 to 22)( days 47-54)
      • transcription factor Msx2 is expressed in interdigital mesenchyme, regulates BMP4-mediated programmed cell death pathway
      • transcription factor Hox-7 also expressed in interdigital zones
  • Limb patterning    
    • Proximodistal 
      • first signaling center to appear is AER
        • controls proximal to distal growth 
        • forms under FGF10 stimulation
      • removal /defect in AER results in proximal limb truncation
        • example is central deficiency (cleft hand)
        • another example is radial clubhand (radial dysplasia, absence of radius)
      • FGFs expressed in AER include FGF4, FGF8, FGF9, and FGF 17
        • FGF8 expressed earliest and is obligatory for normal limb development
        • FGF4, 9 and 17 are redundant
        • disrupted FGF signalling leads to arrested limb development
    • Anteroposterior (radioulnar) limb growth  (nomenclature: ulnar=posterior, radius=anterior)
      • second signaling center to appear is ZPA (zone of polarizing activity), along posterior limb bud  
        • grafting ZPA on anterior limb margin leads to mirror-image digit duplication (ulnar dimelia, or mirror hand)   
      • signaling molecule is Shh compound (dose dependent)
        • normal
          • high concentration of Shh on posterior (ulnar) side for small finger development
          • low concentration of Shh on anterior (radial) side for thumb development
        • posterior/ulnar side abnormalities
          • abnormal upregulation of Shh in the ZPA results in polydactly on the ulnar (posterior) side
            • extent of duplication is dose dependent (higher dose = more replication)
          • downregulation of Shh (on the posterior/ulnar side) leads to loss of ulnar digits
        • anterior/radial side abnormalities
          • abnormal upregulation of Shh in the anterior aspect of the limb bud (where Shh concentration is supposed to be low) leads to loss of thumb  
        • timing
          • posterior elements (little finger/ulna) are formed EARLY prior to anterior elements which are formed LATE (radius/thumb)
          •  disruption of AP patterning will result in loss of later forming elements (radius/thumb)
    • Dorsoventral axis  
      • third signaling center is non-AER limb ectoderm /Wnt signalling center (progress zone, PZ)
      • dorsal limb ectoderm expresses WNT7a 
        • activates Lmx1b (LIM-homeodomain factor) to regulate dorsal patterning
        • WNT7a is responsible for all dorsal features (including nails)
      • ventral ectoderm expresses en-1 (engrailed-1 protein, antagonistic to WNT7a)
        • inhibits WNT7a (and restricts it to dorsal ectoderm)
        • allows ventral limb development
  • Key Genes
    • Sonic Hedgehog (Shh) genes
      • secreted by ZPA
      • involved with HOX gene expression
      • anterior-posterior (radioulnar) growth
        • anterior (radial) mesoderm expresses ALX4
        • posterior (ulnar) mesoderm expresses Hox8
      • concentration gradient dictates formation of digits
        • little finger develops where there is highest Shh concentration
        • thumb develops where there is lowest Shh concentration
      • activates Gremlin
        • Gremlin inhibits BMPs that would otherwise block FGF expression in the AER
    • Hox genes
      • anterior-posterior (radioulnar) patterning
        • together with Shh
      • regulate somatization of the axial skeleton, essentially patterning digit formation 
    • Wnt genes (Wnt7a)
      • expressed in dorsal (non-AER) ectoderm (Wnt signalling center)
      • dorsal-ventral growth
  • Mutations
    • removal of AER
      • truncated limb
    • duplication of ZPA
      • mirror-image duplication of the limb    
Key Genes/Regions
Gene/Region Expresses Regulates
Apical ectodermal ridge (AER) FGF8 is dominant (also FGF 4, 9, 17, which are redundant)
Proximal to distal growth and interdigital necrosis
Zone of polarizing activity (ZPA) Shh
Anterior-posterior (radio-ulnar) growth
Non-AER limb ectoderm (dorsal) Dorsal ectoderm expresses WNT7a, that activates Lmx1b (regulates dorsal patterning) Dorso-ventral growth
Non-AER limb ectoderm (ventral) Ventral ectoderm expresses en-1, antagonistic to WNT7a (regulates ventral patterning) Dorso-ventral growth
Spine and Spinal Cord Development
  • Somites
    • the spinal column originates from pairs of mesodermal structures known as somites 
    • somites develop in a cranial to caudal direction on either side of the notochord and neural tube
      • this process is dependent on the presence of the paraxis gene
    • somite layers
      • sclerotome  
        • layer will become the vertebral bodies and annulus fibrosus 
      • myotome
        • will lead to myoblasts
      • dermatome
        • becomes skin
  • Dorso-vental patterning
    • dorso-vental patterning of the neural tube determined by counteracting activities of
      • Sonic Hedgehog (Shh)
        • in the floor plate and notochord (ventral)  
      • canonical Wnt/β-catenin
        • in the roof plate (dorsal)  
  • Metameric shift phenomenon
    • the phenomenon of how the spinal nerves, which originally ran in the center of the sclerotome, exit between the two vertebral bodies at each level. 
  • Progression
    • neural crest 
      • forms PNS, pia mater, spinal ganglia, sympathetic trunk
    • neural tube  
      • forms spinal cord
    • notochord
      • forms anterior vertebral bodies and nucleus pulposus  
  • Ossification centers
    • vertebrae have 3 primary ossification centers
      • centrum (anterior vertebral body)
      • neural arch (posterior elements, pedicles, small portion of anterior vertebra)
      • costal element (anterior part of lateral mass, transverse process, or rib)
  • Intervertebral disc
    • nucleus pulposus forms from notochord  
    • annulus fibrosus forms from sclerotome  

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