Limb Bud - Position and Formation

Position and Formation

Limb buds form from cells located in the greater limb field region. The Hox genes, which define features along the anterior-posterior axis, dictate at which point along that axis limb buds will form. Though limbs emerge at different locations in different species, their position always correlates with the level of Hox gene expression along the anterior-posterior axis. The upper limb buds, which are destined to become forelimbs (arms in humans), form at a location between the lower cervical vertebrae and upper thoracic vertebrae. The lower limb buds, which will become hindlimbs (legs in humans), form a few days after the upper limb buds near the lumbar vertebrae and upper sacral vertebrae. All limb buds must rely on other signaling factors to obtain their identity. Hox gene expression influences expression of Tbx proteins that in turn dictate limb identity. In humans, Tbx4 specifies hindlimb status, while Tbx5 specifies forelimb status. In mice, however, both hindlimbs and forelimbs can develop in the presence of either Tbx4 or Tbx5. Here, Pitx1 appears to be necessary for specification of the hindlimb, whereas its absence results in forelimb development. Tbx4 and Tbx5 appear to be important for limb outgrowth, but not necessarily their identity in mice. Before limb development begins, FGF10 expression becomes restricted to the proliferating mesenchymal cells of the lateral plate mesoderm, which form the limb bud and become limb mesenchyme. WNT2B and WNT8C stabilize this FGF10 expression in the forelimb and hindlimb, respectively. This FGF10 expression stimulates WNT3 expression in the above ectodermal cells – resulting in formation of the apical ectodermal ridge (AER) as well as inducing FGF8 expression. The FGF8 secreted by the AER acts to keep the cells of the limb mesenchyme in a mitotically active state and sustains their production of FGF10. This positive feedback loop between the limb mesenchyme and the AER maintains the continued growth and development of the entire limb. In addition to limb outgrowth, the formation of a crucial signaling center, the zone of polarizing activity (ZPA), in a small posterior portion of the limb bud helps to establish anterior-posterior polarity in the limb through secretion of the protein Sonic hedgehog (Shh). The ZPA also plays an important role in initially specifying digit identity, while later maintaining proper AER morphology and continued FGF8 secretion – to ensure proper mitotic activity of the limb bud mesenchyme beneath. Eventually, the limb mesenchyme will form the bones, muscles, and other connective tissue of the limb.