Starts in: 4^th week (Upper limb initiated 3-4 days before and stays ahead of the lower limb)
Finished by: 8^th week

1. Form from somatic lateral plate mesoderm
2. Edge of bud the apical ectodermal ridge (AER)→critical for limb formation
3. Within the limb bud are the progress zone (high proliferation) and the mesenchymal mesoderm (stop dividing, differentiate into proper proximal/distal fate)

1. Intermediate mesoderm→ FGF8 (mitogen)→ ↑ LPM to proliferate→ Induces Limb bud
2. FGF8→initiates Apical Ectodermal Ridge (AER)
3. AER releases FGF8→ stimulates growth in progress zone→ controls where the limb forms along the A/P axis of the body

Limb axes: Limb axes are unusual: anterior (thumb), dorsal (back of the hand), distal (fingers)

Development of the three limb axes
Proximal/Distal Axis	AER (apical ectodermal ridge) formed by ectodermal thickening Influences underlying mesenchyme to proliferate (progress zone) Mesenchyme differentiates first furthest from AER Control of P/D differentiation: Pattern of HOX genes (more distally)
2. Anterior/Posterior Axis	Control of A/P patterning: zone of polarizing activity (ZPA) is the mesenchymal tissue in the posterior limb bud is responsible Shh gradient: ZPA produces Shh which acts as a morphogen Gli3 repressor gradient (highest in anterior) → Gli is cleaved into the active form in absence of Shh Shh receptor (Ptc) is also distributed in a graded manner
3. Dorsal/Ventral Axis	Wnt-7a from dorsal limb bud ectoderm→ induces Lmx-1 in dorsal limb mesenchyme,→dorsalizes limbOn the ventral side: EN-1 (from ectoderm) represses WNT-7a expression → hence Lmx-1 is inactive and the limb is centralized

The pattern signaling of Shh, Wnt-7a, and FGF are codependent and integrated
Forelimb/hindlimb:

1. Leg expresses: Pitx1 and Tbx4 txn factors (controlled by Hox-c genes)
2. Arm expresses: Tbx5 which inhibits Tbx4

Digits formation:
1. AER delineates where digits will form.
2. ZPA & SHH determine which digits will form & where.
3. The webbing of finger and toes removed by apoptosis induced by BMP
Limb bud malformations often related to urogenital malformations: both involve intermediate mesoderm defects.

Cells differentiate first furthest from AER
Endochondral ossification: Mesenchyme differentiates→ cartilage→ bone
Lateral plate mesoderm condenses along long axis→ cartilaginous perichondrium→ replaced by bone
Interzones between bones: differentiation into joint tissue

Ossification:
- Diaphyses ossified by birth (begins at 7^th week)
- Epiphyses shortly thereafter
- Continued bone growth occurs at the epiphyseal growth plate

Connective tissues dictate the pattern of muscle formation by passively guiding the direction of migration of muscle precursor cells
6^th week: connective tissue guides the migration of muscle cell precursors from somite to limb bud → forming dorsal/ventral condensations
Mesodermal Condensations differentiate into →myoblasts (muscle precursors)
Dorsal and ventral muscle masses differentiate

	Dorsal muscle mass	Ventral muscle mass
1. Nerve supply	Extensors and abductors (supinators); supplied by dorsal (posterior) branches of ventral primary rami.	Flexors and adductors (protonators); supplied by ventral (anterior) branch of ventral primary rami
2. Upper limb	Posterior compartment (arm); posterior & lateral compartment (forearm); deltoid, latissimus dorsi, rhomboids, levator scapulae, serratus anterior, teres major/minor, subscapularis	Anterior compartment (arm & forearm); palmar surface of hand
3. Lower Limb	Anterior compartment (thigh); anterior & lateral compartment (leg); tensor fascia lata, short head of biceps femoris, dorsum of foot, gluteus max/med/min, piriformis, iliacus, psoas	Medial & posterior compartments (thigh) [Except short head of biceps], posterior compartment (leg), plantar surface of foot, obturator internus, gemellus superior/inferior, quadratus femoris

Upper limbs	Lower limbs
Upper limbs from brachial plexus (C5-T1),	lower limbs from lumbosacral plexus (L4-S3)
Dorsal (posterior): radial and axillary nerves [extend & supinate]Ventral (anterior): median, ulnar, musculocutaneous [flex & protonate]	Dorsal (posterior): femoral, fibular, & gluteals [extend & abduct]Ventral (anterior): tibial, obturator, nn to short rotators [flex & adduct]

Dorsal branches innervate dorsal muscle mass; ventral branches innervate ventral muscle mass
Axon growth guidance by mesenchyme controls the final pattern of innervation
Postganglionic sympathetic fibers use developing vasculature as a guide
Sensory axons develop later (slower) via sympathetic and motor axons as a guide

1. Amelia	Early loss of FGF signaling: absence of an entire limb
2. Meromelia	Later or partial loss of FGF signaling: absence of part of a limb
3. Phocomelia	Partial loss of FGF or HOX disruption short→ poorly formed limb P/D axis defects: Early teratogen → usually limb bud initiation or limb bud elongation defects; earlier time point→ more severe deformity (limb truncations)
4. Ectrodactyly	A/P axis defects: too few digits (AER too narrow/deficient)
5. Polydactyly	A/P axis defects: too many digits (AER too broad)
6. Syndactyly	Defects (MSX2, BMP4) → Soft tissue fusion between digits due (fused digital soft tissue) to lack/insufficient of normal programmed cell death (apoptosis) within the interdigital regions
7. Mirror Image Duplications	A/P axis defects: occurs via duplication of ZPA