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| Apical ectodermal ridge | |
|---|---|
 The apical ectodermal ridge is a region of thickened epithelium at the most distal end of the limb bud. The zone of polarising activity (ZPA) is at the posterior part of the limb bud. | |
| Details | |
| Identifiers | |
| Latin | crista ectodermalis apicalis |
| TE | ectodermal ridge_by_E5.0.3.0.0.3.4 E5.0.3.0.0.3.4 |
| Anatomical terminology | |
Theapical ectodermal ridge (AER) is a structure that forms from theectodermal cells at the distal end of eachlimb 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 limbmesenchyme—including thezone of polarizing activity (ZPA)—continue to communicate with each other to direct furtherlimb development.[1]
The position of the limb bud, and hence the AER, is specified by the expression boundaries ofHox genes in the embryonic trunk. At these positions, the induction of cell outgrowth is thought to be mediated by apositive feedback loop offibroblast growth factors (FGFs) between theintermediate mesoderm, thelateral plate mesoderm and thesurface ectoderm.FGF8 in the intermediate mesoderm signals to the lateral mesoderm, restricting the expression ofFGF10 through intermediateWnt signals. Then, FGF10 in the lateral plate mesoderm signals to the surface ectoderm to create the AER, which expresses FGF8.[2]
The AER is known to expressFGF2,FGF4,FGF8, andFGF9, while the limb bud mesenchyme expressesFGF2 andFGF10. Embryo manipulation experiments have shown that some of these FGFs alone are sufficient for mimicking the AER.[3]
Morphologically, the AER emerges as a thickening of the ectoderm at the distal rim of the limb bud. This distinct structure runs along the anterior-posterior axis of the limb bud and subsequently separates the dorsal side of the limb from its ventral side.
In the wing bud in chick embryos, the AER becomes anatomically distinguishable at the late stage of development18HH (corresponding to 3 day-old embryos), when the distal ectodermal cells of the bud acquire a columnar shape distinguishing them from thecuboidal ectoderm. At stage20HH (corresponding to 3.5 day-old embryos), the AER appears as a strip ofpseudostratified epithelium which is maintained until 23-24HH (corresponding to 4-4.5 day-old embryos). Afterwards, the AER progressively decreases in height and eventually regresses.[4]
In mouse embryos, the ventral ectoderm of the emerging forelimb at E9.5 (embryonic day 9.5[5]) already appears thicker in comparison to the dorsal ectoderm and it corresponds to the early AER.[6][7] By E10, this thickening is more noticeable since the epithelium now consists of two layers and becomes confined to the ventral-distal margin of the bud although it is not detectable in living specimens using light microscope or byscanning electron microscopy (SEM).[8] Between E10.5-11, a linear and compact AER with a polystratified epithelial structure (3-4 layers) has formed and positioned itself at the distal dorso-ventral boundary of the bud.[6][8][9][10] After reaching its maximum height, the AER in mouse limb buds flattens and eventually become indistinguishable from the dorsal and ventral ectoderm.[8][11][12] The structure of the human AER is similar to the mouse AER.[13]
In addition to wings in chicks and forelimbs in mice, pectoral fins in zebrafish serve as a model to study vertebrate limb formation. Despite fin and limb developmental processes share many similarities,[14] they exhibit significant differences, one of which is the AER maintenance. While in birds and mammals the limb AER persists until the end of digit-patterning stage and eventually regresses, the fin AER transforms into an extended structure, named theapical ectodermal fold (AEF).[15] After the AER-AEF transition at 36 hours post fertilization, the AEF is located distal to the circumferential blood vessels of the fin bud. The AEF potentially functions as an inhibitor to fin outgrowth since removing the AEF results in the formation of a new AER and subsequently a new AEF. In addition, repeated AF removal leads to excessive elongation of the fin mesenchyme, potentially because of prolonged exposure of AER signals to the fin mesenchyme.[16] Recently, the AER, which has long been thought to consist of only ectodermal cells, in fact composes of both mesodermal and ectodermal cells in zebrafish.[17]
Associated molecules include:[1]
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.[1]
The AER acts to:[1]
TheHox 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-secretedFGFs and ZPA-secretedShh 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 inproximal-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 themesoderm is primed to respond to it differently. These three phases of regulation reveal a mechanism by whichnatural selection can independently modify each of the three limb segments – thestylopod, thezeugopod, and theautopod.[18]
The Hox genes are physically linked in four chromosomal clusters (Hoxa, Hoxb, Hoxc, Hoxd),[18] 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 withHOXB9, 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 andHOXD 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. WhereasHOXC andHOXB genes tend to be restricted to specific limbs,HOXA andHOXD are usually expressed in all limbs.HOXD9 andHOXD10 are expressed in the developing limb throughout the entire anterior-posterior axis, followed byHOXD11,HOXD12,HOXD13, which are each expressed in more posterior regions, withHOXD13 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.[18]
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.)
The precise microenvironmental cues present at the D-V boundary are crucial for AER formation. When the limb bud is dorsalized - inlimbless mutants, for example - and no dorsal-ventral boundary exists, the AER is unable to form and limb development halts.
The removal of the AER results in truncated limbs where only thestylopod is present.[20] The transplantation of an additional AER results in the duplication of limb structures, usually as a mirror image next to the already developing limb. The mirror image reflection is a result of the transplanted AER obeying signals from the existing ZPA.
Implantation of a plastic bead soaked in FGF-4 or FGF-2 will induce formation of a limb bud in an embryo, but proliferation will cease prematurely unless additional beads are added to maintain appropriate levels of the FGF. Implantation of sufficient beads can induce formation of a 'normal' additional limb at an arbitrary location in the embryo.[21][22]
Transplantation of the AER to flank mesoderm between the normal limb buds results inectopic limbs. If the AER is transplanted closer to theforelimb bud, the ectopic limb develops like a forelimb. If the AER is transplanted closer to the hindlimb bud, the ectopic limb develops like ahindlimb.[23] If the AER is transplanted near the middle, the ectopic limb has both forelimb and hindlimb features.[24]
Transplantation of an AER that would give rise to an arm (or wing, as these experiments are commonly performed on chicken embryos) to a limb field developing into a leg does not produce an arm and leg at the same location, but rather two legs. In contrast, transplantation of cells from the progress zone of a developing arm to replace the progress zone of a developing leg will produce a limb with leg structures proximally (femur,knee) and arm structures distally (hand,fingers). Thus it is the mesodermal cells of the progress zone, not the ectodermal cells of the AER, that control the identity of the limb.[25]
AER timing does not regulate the fate specification of the underlying mesoderm, as shown by one set of experiments. 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 zoneis 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).[20]