The limb bud is a structure formed early in vertebrate limb development. As a result of interactions between the ectoderm and underlying mesoderm, formation occurs roughly around the fourth week of development. In the development of the human embryo the upper limb bud appears in the third week and the lower limb bud appears four days later. The limb bud consists of undifferentiated mesoderm cells that are sheathed in ectoderm. As a result of cell signaling interactions between the ectoderm and underlying mesoderm cells, formation of the developing limb bud occurs as mesenchymal cells from the lateral plate mesoderm and somites begin to proliferate to the point where they create a bulge under the ectodermal cells above. The mesoderm cells in the limb bud that come from the lateral plate mesoderm will eventually differentiate into the developing limb's connective tissues, such as cartilage, bone, and tendon. Moreover, the mesoderm cells that come from the somites will eventually differentiate into the myogenic cells of the limb muscles. The limb bud remains active throughout much of limb development as it stimulates the creation and positive feedback retention of two signaling regions: the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) with the mesenchymal cells. These signaling centers are crucial to the proper formation of a limb that is correctly oriented with its corresponding axial polarity in the developing organism. Research has determined that the AER signaling region within the limb bud determines the proximal-distal axis formation of the limb using FGF signals. ZPA signaling establishes the anterior-posterior axis formation of the limb using Shh signals. Additionally, though not known as a specific signaling region like AER and ZPA, the dorsal-ventral axis is established in the limb bud by the competitive Wnt7a and BMP signals that the dorsal ectoderm and ventral ectoderm use respectively. Because all of these signaling systems reciprocally sustain each other's activity, limb development is essentially autonomous after these signaling regions have been established.
Position and formation The Hox genes, which define features along the anterior-posterior axis of a developing organism, determine at which points along the axis that limb buds will form. Though limbs emerge at different locations in different species, their positions always correlate with the level of Hox gene expression along the anterior-posterior axis. All limb buds must also rely on other signaling factors to obtain their forelimb or hindlimb identity; Hox gene expression influences expression of T-box proteins that, in turn, determine limb identity for certain organisms. In turn, the activation of T-box protein activates signaling cascades that involve the Wnt signaling pathway and FGF signals. Before limb development begins, T-box proteins initiate FGF10 expression in the proliferating mesenchymal cells of the lateral plate mesoderm, which form the limb bud mesoderm. 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 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. positive feedback loop between the limb mesenchymal cells 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. In chickens, 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. In fact, it is the Pitx1 and Pitx2 genes that appears to be necessary for specification of the developing hindlimb, whereas their absence results in forelimb development.Tbx4 and Tbx5 appear to be important specifically for limb outgrowth in mice.
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