Apical ectodermal ridge
|Apical ectodermal ridge|
|The apical ectodermal ridge is a thickened epithelium at the most distal end of the limb bud. The zone of polarizing activity is at the posterior part of the limb bud.|
|Latin||crista ectodermalis apicalis|
Apical Ectodermal Ridge (AER)
The apical ectodermal ridge (AER) is a structure that forms from the ectodermal cells at the distal end of each limb bud and acts as a major signaling center to ensure proper development of a limb. After the limb bud induces AER formation, the AER and limb mesenchyme – including the zone of polarizing activity (ZPA) - continue to communicate with each other to direct further limb development.
FGF10 secretions from the mesenchyme cells of the limb field interact with the ectodermal cells above, and induce the formation of the AER on the distal end of the developing limb. The presence of a dorsal-ventral ectodermal boundary is crucial for AER formation – the AER can only form at that divide.
- Maintain the limb mesenchyme in a mitotically active state and focused on its task – the distal outgrowth of the limb. This is achieved by secretion of FGF8, which signals the limb mesodermal cells to continue proliferation, and secreting FGF10, which winds up maintaining the AER.
- Sustain expression of the molecules that establish the anterior-posterior axis. The FGFs secreted by the AER act upon the mesenchyme cells – including the zone of polarizing activity (ZPA). Thus, the AER causes the ZPA to continue secreting Sonic hedgehog (Shh), which is involved with Hox gene expression in establishing the anterior-posterior polarity in the developing limb. Shh also activates Gremlin, which inhibits bone morphogenetic proteins (BMPs) that would normally block FGF expression in the AER. In this manner, the ZPA and AER sustain each other through a positive feedback loop involving FGFs, Shh, and Gremlin.
- Communicate with the proteins that determine the anterior-posterior and dorsal-ventral axes to supply instructions concerning differentiation and cell fates. The FGFs secreted by the AER interact with the limb mesenchyme – including the ZPA – to induce further FGF and Shh expression. These signals then regulate Hox gene expression, which influence differentiation activity and determines what phenotypes the cells will adopt. The secreted Shh also activates Gremlin, which inhibits members of the BMP family. BMPs inhibit FGF expression in the AER, so the FGF secreted by the AER ends up providing feedback (via Shh and Gremlin) that will dictate cellular differentiation involved in sculpting the limb.
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 differently to it. 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.
Relevant Experiments 
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.
- When the AER from a late limb bud is transplanted to an earlier limb bud, the limb forms normally. The converse – transplantation of an early limb bud to a late limb bud – also results in normal limb development. However, the underlying mesoderm in the progress zone ‘’is’’ fate specified. If progress zone mesoderm is transplanted along with the AER, then additional finger/toes are formed (for an early-->late transplantation) or the finger/toes are formed too early (for a late-->early transplantation).
- AER Formation Relies on Dorsal-Ventral Boundary:
The Precise Microenvironmental Cues present at the D-V boundary are crucial for AER formation
- When the limb bud is dorsalized - in limbless mutants, for example - and no dorsal-ventral boundary exists, the AER is unable to form and limb development halts.
- 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.
- 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).