Limb development is a complex process involving multiple signaling centers and molecular pathways. It starts with limb bud formation and progresses through stages of growth, patterning, and tissue differentiation along three axes.

Key players in limb development include the Apical Ectodermal Ridge (AER) and Zone of Polarizing Activity (ZPA). These signaling centers coordinate growth and patterning through molecules like FGFs and Sonic hedgehog, shaping the final limb structure.

Limb Development Stages

Limb Bud Formation and Elongation

• Limb development initiates with formation of limb buds, outgrowths of mesenchymal cells covered by ectoderm
• Limb bud elongates along proximal-distal axis, proximal region develops first followed by distal region
• Cartilage condensation occurs within limb bud, forming initial skeletal elements that later ossify into bones
• Limb undergoes patterning along three axes
  • Proximal-distal axis (shoulder to fingertips)
  • Anterior-posterior axis (thumb to pinky)
  • Dorsal-ventral axis (back of hand to palm)

Tissue Differentiation and Morphogenesis

• Interdigital apoptosis separates developing digits and shapes final limb structure
• Muscle precursor cells migrate into limb bud from somites and differentiate into specific muscle groups (biceps, triceps)
• Vascularization and innervation of developing limb occur concurrently with skeletal and muscular development
  • Blood vessels form to supply nutrients and oxygen
  • Nerves grow into limb to provide motor control and sensory input

AER and ZPA Roles in Patterning

Apical Ectodermal Ridge (AER) Function

• AER forms as thickened region of ectoderm at distal tip of limb bud, directing proximal-distal outgrowth
• Secretes fibroblast growth factors (FGFs) maintaining underlying mesenchyme in proliferative state
  • FGF8 and FGF10 are key signaling molecules
• AER removal results in truncated limbs (chicken wing experiments)
• Grafting additional AER tissue induces formation of supernumerary limb structures (extra digits)

Zone of Polarizing Activity (ZPA) Function

• ZPA comprises group of mesenchymal cells in posterior region of limb bud, establishing anterior-posterior axis
• Secretes Sonic hedgehog (Shh), forming concentration gradient across limb bud to specify digit identity
  • High Shh levels lead to posterior digit formation (pinky)
  • Low Shh levels result in anterior digit formation (thumb)
• ZPA transplantation to anterior region of host limb bud induces mirror-image digit duplications (classic experiment by Saunders and Gasseling)

AER-ZPA Feedback Loop

• AER and ZPA maintain each other through positive feedback loop involving FGF and Shh signaling
• FGFs from AER maintain Shh expression in ZPA
• Shh from ZPA promotes FGF production in AER
• This feedback loop ensures coordinated growth and patterning along both proximal-distal and anterior-posterior axes

Molecular Mechanisms in Limb Axis Formation

Proximal-Distal Axis Establishment

• Proximal-distal axis established by gradient of retinoic acid (RA) from body wall and FGFs from AER
• RA promotes expression of proximal genes like Meis1/2
  • Meis1/2 specify proximal limb structures (upper arm/leg)
• FGFs induce distal gene expression such as Hoxa13 and Hoxd13
  • Hoxa13 and Hoxd13 specify distal structures (hands/feet)
• Two-signal model explains progressive specification of limb segments
  • Early exposure to both RA and FGF specifies proximal structures
  • Later exposure to only FGF specifies distal structures

Anterior-Posterior Axis Patterning

• Anterior-posterior axis patterned by gradient of Sonic hedgehog (Shh) secreted from ZPA
• Shh induces expression of Gli transcription factors, regulating target genes in concentration-dependent manner
  • Gli1 and Gli2 act as activators, promoting posterior fates
  • Gli3 acts as repressor, promoting anterior fates
• Hand2 expressed in posterior limb bud, essential for ZPA formation and Shh expression
• Gli3 repressor forms opposing gradient to Shh, promoting anterior fates in limb bud
• Hox genes, particularly HoxA and HoxD clusters, expressed in nested patterns along both axes
  • Hoxd9-Hoxd13 progressively expressed from anterior to posterior
  • Hoxa11 and Hoxa13 expressed in proximal and distal regions, respectively

Cell-Cell Interactions in Limb Patterning

Signaling Pathways in Limb Development

• Reciprocal signaling between AER and underlying mesenchyme maintains limb bud outgrowth through FGF-Shh feedback loop
• Wnt signaling from AER crucial for maintaining AER and promoting limb outgrowth
  • Wnt3a and Wnt7a are key players
• Bone morphogenetic proteins (BMPs) regulate AER formation, maintenance, and interdigital apoptosis
  • BMP2 and BMP4 promote cell death between developing digits
• Notch signaling regulates AER formation, maintenance, and digit formation
  • Notch1 and its ligands (Delta, Jagged) involved in boundary formation

Dorsal-Ventral Axis Establishment

• Dorsal-ventral axis established by Wnt7a expression in dorsal ectoderm and En1 expression in ventral ectoderm
• Lmx1b, induced by Wnt7a, specifies dorsal mesenchyme identity
  • Lmx1b mutations lead to nail-patella syndrome in humans

Cell Adhesion and Tissue Organization

• Ephrin signaling involved in segregation of cartilage elements and patterning of limb musculature
  • EphA4 and ephrin-A5 guide muscle precursor cell migration
• Cell adhesion molecules, such as N-cadherin, play important roles in mesenchymal condensation during cartilage formation
  • N-cadherin mediates cell-cell adhesion in pre-cartilage condensations