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|lateral plate mesoderm|
The limb bud is a structure formed early in limb development. As a result of interactions between the ectoderm and underlying mesoderm, formation occurs roughly around the fourth week of development  as mesenchymal cells from the lateral plate mesoderm and the somites begin to proliferate to the point where they create a bulge under the ectodermal cells above. The limb bud remains active throughout much of limb development, and its signaling stimulates formation of another signaling center, the apical ectodermal ridge (AER) as well as formation of the zone of polarizing activity (ZPA) within the mesenchyme. The mesenchymal cells of the limb bud, which stimulate AER formation as well as maintain AER activity, determine what type of limb will form. ZPA signaling will establish polarity of the limb, as well as sustain proper AER activity.
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.
Relationship between hox gene expression and limb patterning
The Hox genes, which initially establish the anterior-posterior axis of the entire embryo, continue to participate in the dynamic regulation of limb development even after the AER and ZPA have been established. Complex communication ensues as AER-secreted FGFs and ZPA-secreted Shh initiate and regulate Hox gene expression in the developing limb bud. Though many of the finer details remain to be resolved, a number of significant connections between Hox gene expression and the impact on limb development have been discovered. The pattern of Hox gene expression can be divided up into three phases throughout limb bud development, which corresponds to three key boundaries in proximal-distal limb development. The transition from the first phase to the second phase is marked by the introduction of Shh from the ZPA. The transition into the third phase is then marked by changes in how the limb bud mesenchyme responds to Shh signaling. This means that although Shh signaling is required, its effects change over time as the mesoderm is primed to respond to it differently. These three phases of regulation reveal a mechanism by which natural selection can independently modify each of the three limb segments – the stylopod, the zeugopod, and the autopod. The Hox genes are “physically linked in four chromosomal clusters (Hoxa, Hoxb, Hoxc, Hoxd), and their physical position on the chromosome seems to correlate with the time and place of expression. For example, the most 3’ Hoxc genes (HOXC4, HOXC5) are expressed only in the anterior limbs (wings) in chickens, while the more 5’ genes (HOXC9, HOXC10, HOXC11) are expressed only in the posterior limbs (legs). The intermediate genes (HOXC6, HOXC8) are expressed in both the upper and lower limbs. Within the limb bud, expression also varies as a function of the position along the anterior-posterior axis. Such is the case with HOXB9, which is most highly expressed next to the AER, and decreases when moving anteriorly to posteriorly, resulting in the least HOXB9 expression next to the posterior ZPA. HOXB9 expression is inversely proportional to the level of Shh expression, which makes sense, as the ZPA secretes Shh. HOXA and HOXD genes for the most part follow nested expression domains, in which they are activated uniformly along the anterior-posterior axis of the limb itself, but not the anterior-posterior axis of the entire body. Whereas HOXC and HOXB genes tend to be restricted to specific limbs, HOXA and HOXD are usually expressed in all limbs. HOXD9 and HOXD10 are expressed in the developing limb throughout the entire anterior-posterior axis, followed by HOXD11, HOXD12, HOXD13, which are each expressed in more posterior regions, with HOXD13 being restricted to only the most posterior regions of the limb bud. As a result, HOXD expression clusters around the posterior ZPA (where HOXD9, 10, 11, 12, and 13 are all expressed), while less expression occurs around the AER, where only HOXD9 and HOXD10 are expressed.
- FGF10 can induce limb formation, but Tbx, Pitx, and Hox determine identity
By mimicking the initial FGF10 secretions of the lateral plate mesoderm cells, limb development can be initiated. Other signaling molecules are implicated in determining the limb's identity.
- Placement of FGF10-containing beads beneath chick ectodermal cells results in the formation a limb bud, AER, ZPA and, subsequently, an entire limb. When the beads created limb buds towards the anterior region, forelimb formation coincided with Tbx5 expression, while hindlimb formation coincided with Tbx4 expression. When beads were placed in the middle of the flank tissue, the anterior portion expressed Tbx5 and forelimb features, while the posterior portion of the limb expressed Tbx4 and hindlimb features.
- When chick embryos were engineered to constitutively express Tbx4 (via viral-transfection) throughout their flank tissue, every limb they grew was a leg, even those that formed in the anterior region, which would normally become wings. This confirms the role of Tbx proteins in the type of limb that develops.
- Knocking out Tbx4 or Tbx5 knockout prevents FGF10 expression in the lateral plate mesoderm in mice.The Hox pathway may affect Tbx expression, which in turn affects FGF10 expression.
- When Pitx1 was incorrectly expressed in mouse forelimbs, several hindlimb-associated genes (Tbx4, HOXC10) were turned on and drastic alterations of the muscles, bones, and tendons shifted the phenotype towards that of a hindlimb. This indicates that Pitx1 – through Tbx4 - plays a role in the emergence of hindlimb properties.
- HOXD11 expression correlates with Shh secretion
HOXD11 is expressed most posteriorly, near the ZPA, where the highest levels of Shh expression occur.
- When retinoic acid is applied to induce Shh expression, a ZPA is transplanted, or ectopic expression of Shh is stimulated, HOXD11 expression follows.
- Mesenchyme cells determine limb identity, but the AER maintains limb outgrowth through FGF secretion
These experiments reveal that the limb mesenchyme contains the necessary information concerning limb identity, but the AER is needed to stimulate the mesenchyme to live up to its destiny (of becoming an arm, leg, etc.)
- When the AER is removed, limb development halts. If an FGF bead is added in the AER’s place, normal limb development proceeds.
- When an extra AER is added, two limbs form.
- When forelimb mesenchyme is replaced with hindlimb mesenchyme, a hindlimb grows.
- When forelimb mesenchyme is replaced with non-limb mesenchyme, the AER regresses, and limb development halts
- ZPA's role in establishing polarity and further limb development
The ZPA first specifies anterior-posterior polarity (and dictates digit identity), and then, by sustaining AER activity, it ensures that the necessary cell proliferation occurs for normal formation of a five-digit limb.
- When Shh normally secreted from the ZPA is inhibited (either through use of tamoxifen or Shh-null mutants) the AER morphology, particularly its anterior extent, is perturbed and its FGF8 signaling decreased. As a result of Shh downregulation during limb bud expansion, the number of digits was decreased, but the identities of the formed digits was not altered.
Associated molecules include:
- FGF10 = Initially, Tbx proteins induce secretion of FGF10 by cells in the lateral plate mesoderm. Later, FGF10 expression is restricted to the developing limb mesenchyme, where it is stabilized by WNT8C or WNT2B. FGF10 expression activates secretion of WNT3A, which acts upon the apical ectodermal ridge (AER) and induces FGF8 expression. The mesenchyme, through FGF10 secretion, is involved in a positive feedback loop with the AER, through FGF8 secretion.
- FGF8 = Secreted by the apical ectodermal ridge cells. Acts upon the mesenchyme cells, to maintain their proliferative state. Also induces the mesenchymal cells to secrete FGF10, which acts through WNT3A to sustain the AER’s expression of FGF8.
- WNT3A = Acts as a middle man in the positive feedback loop between the AER and limb mesenchyme. Activated by FGF10 expression, activates FGF8 expression.
- Shh = Secreted by the ZPA in the limb bud mesenchyme. Creates concentration gradient that dictates formation of the five distinct digits. Digit 5 (pinkie) results from exposure to high Shh concentrations, while digit 1 (thumb) on the opposite end of the spectrum develops in response to low concentrations of Shh. Shh expression has been shown in many, but not all circumstances, to be heavily connected with Hox gene expression. Shh also (via Gremlin) blocks bone morphogenic protein (BMP) activity. By blocking BMP activity, FGF expression in the AER is maintained.
- Tbx4/5 = Involved with the development of hindlimbs versus forelimbs. Though in chicks, they seem to be the primary factors involved in limb identity, in mice it appears that Tbx4 is merely a downstream messenger enforcing the hindlimb-forming instructions of Pitx1. Whether Pitx1 merely diverts a prospective forelimb from that path to become a hindlimb, or if Tbx5 is activated by another Pitx1-like messenger, is unknown.
- Pitx1 = Responsible for the development of a hindlimb-associated phenotype. Tbx4 is one of its downstream targets
- Hox genes =  Responsible for dictating the anterior-posterior axis of an organism, and Is intricately involved in patterning of the developing limb in conjunction with Shh. Influences the activity of TBX and FGF (and possibly Pitx1) proteins. Determines where limb buds will form, and what limbs will develop there.
- Gilbert, Scott F. "Developmental Biology". 9th ed., 2010
- C. E. Nelson et al. "Analysis of Hox gene expression in the chick limb bud" Development. 122, 1449 (1996). 
- Zhu, J., et al. Uncoupling Sonic hedgehog control of pattern and expansion of the developing limb bud. Dev. Cell 14, 624–632 (2008).