Limb Formation L18

• Limb buds consist of undifferentiated mesenchyme enclosed by ectoderm.
• Limb field defines the region of cells capable of forming the limb bud.
• Limb bud development depends on the Apical Ectodermal Ridge (AER), the Zone of Polarizing Activity (ZPA), and various gene signals.
• Mesenchyme cells proliferate from the somatopleuric lateral plate mesoderm into limb skeletal precursors.
• Somites become limb muscle precursors.
• Neural crest cells form pigment cells and sensory axons.

Limb Bud Formation

• Cells gather under the ectoderm to form the limb bud bulge.
• The Apical Ectodermal Ridge (AER) is formed by ectodermal cells at the distal end of each limb bud.
• AER acts as a major signaling center for proper limb development.
• Mesenchyme cells entering the limb field secrete FGF10, inducing the overlying ectoderm to differentiate into a ridge-like thickening and then secrete FGF8.
• FGF8 facilitates the formation of AER and maintains the region by regulating transcription factors for forming different limbs.

Role of the AER

• AER maintains the limb mesenchyme in a mitotically active state, focused on distal outgrowth.
• AER sustains expression of molecules to establish the anterior-posterior axis including:
  • Hox genes which drive production of the limb.
  • Gremlin which indicates limb growth.
  • BMPs such as BMP4, which inhibit FGF to stop limb growth.
• AER maintains limb outgrowth, while mesenchyme cells determine identity.
• Bones are formed via endochondral ossification of mesodermal condensations.
• Muscles form as somitic mesoderm invades the limb bud and differentiates into myoblasts.
• Migrating neural crest cells invade the limb bud to become sensory axons and pigment cells
• Motor axons extend from spinal cord neurons to the limb bud.

Fore and Hind Limb

• Forelimbs express the Tbx5 transcription factor.
• Hindlimbs express the Tbx4 transcription factor.

Proximal-Distal Axis

• Hox genes regulate the proximal-distal axis (shoulder to finger).
• Cells leaving the AER determine their position, with proximal structures developing first (Progress Zone Model).
• The longer a cell spends in the PZ, the more mitoses achieved, and the more distal its specification becomes.
• Cells leaving first form the upper arm.
• Intermediate cells form the forearm.
• The last cells to leave form the hand.

Anterior-Posterior Axis

• Shh protein regulates the anterior-posterior axis (thumb to pinky).
• Shh sustains BMP-2 and BMP-7 proteins, which create the digits.

Dorsal-Ventral Axis

• Wnt7a regulates the dorsal-ventral axis (knuckle to palm).
• Wnt7a is expressed only in the dorsal ectoderm of the limb bud.
• Wnt7a induces activation of the Lmx1 gene in the dorsal mesenchyme.
• Lmx1 encodes a transcription factor required for dorsal cell fates.

Digit Formation

• Cell death is vital for sculpting the limb, via apoptosis.
• Areas sculpted by cell death include:
  • Interdigital necrotic zone to separate digits.
  • Interior necrotic zone to separate ulna and radius.
  • Anterior and posterior necrotic zones for further sculpting.
• BMP proteins (2, 4, 7) signal for cell death and are expressed in the interdigital mesenchyme.
• BMP is expressed between the digits, causing apoptosis between them.

Joint Formation

• BMP induces mesenchymal cells to undergo apoptosis or become cartilage-producing chondrocytes, depending on the developmental stage.
• BMP proteins express at regions between bones where joints will form.
• Cartilage at the end of bones, called epiphyseal growth plates allow the bone to continue to grow as long as epiphyseal growth plates continue to produce chondrocytes.

Limb Anomalies

• Limb anomalies affect 3% of newborns in humans.
• 40-60% of the causes are unknown.
• 20-25% are multifactorial, involving minor genetic anomalies and environmental risk factors.
• 10-13% are environmental.
• 12-25% are due to mutations.

Reduction Defects

• Meromelia: absence of part of a limb.
• Amelia: absence of an entire limb.
• Phocomelia: absence of the proximal limb.

Ectrodactylyl

• Ectrodactylyl is the absence of fingers and toes.
• Lobster claw is where the third digit is missing, and the thumb and index are fused, and has genetic origins.
• Adactylyl: absence of all digits on a limb.