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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.[1]

The position of the limb bud, and hence the AER, is specified by the expression boundaries of Hox genes in the embryonic trunk. At these positions, the induction of cell outgrowth is thought to be mediated by a positive feedback loop of fibroblast growth factors (FGFs) between the intermediate mesoderm, the lateral plate mesoderm and the surface ectoderm. FGF8 in the intermediate mesoderm signals to the lateral mesoderm, restricting the expression of FGF10 through intermediate Wnt 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 express FGF2, FGF4, FGF8, and FGF9, while the limb bud mesenchyme expresses FGF2 and FGF10. Embryo manipulation experiments have shown that some of these FGFs alone are sufficient for mimicking the AER.[3]

Structure

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 development 18HH (corresponding to 3 day-old embryos), when the distal ectodermal cells of the bud acquire a columnar shape distinguishing them from the cuboidal ectoderm. At stage 20HH (corresponding to 3.5 day-old embryos), the AER appears as a strip of pseudostratified 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 by scanning 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 the apical 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]<

The position of the limb bud, and hence the AER, is specified by the expression boundaries of Hox genes in the embryonic trunk. At these positions, the induction of cell outgrowth is thought to be mediated by a positive feedback loop of fibroblast growth factors (FGFs) between the intermediate mesoderm, the lateral plate mesoderm and the surface ectoderm. FGF8 in the intermediate mesoderm signals to the lateral mesoderm, restricting the expression of FGF10 through intermediate Wnt 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 express FGF2, FGF4, FGF8, and FGF9, while the limb bud mesenchyme expresses FGF2 and FGF10. 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 development 18HH (corresponding to 3 day-old embryos), when the distal ectodermal cells of the bud acquire a columnar shape distinguishing them from the cuboidal ectoderm. At stage 20HH (corresponding to 3.5 day-old embryos), the AER appears as a strip of pseudostratified 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 by scanning 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

In the wing bud in chick embryos, the AER becomes anatomically distinguishable at the late stage of development 18HH (corresponding to 3 day-old embryos), when the distal ectodermal cells of the bud acquire a columnar shape distinguishing them from the cuboidal ectoderm. At stage 20HH (corresponding to 3.5 day-old embryos), the AER appears as a strip of pseudostratified 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 by scanning 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 the apical 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: 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 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]

    Function

    The AER acts to:[1]

    • 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.

    Relationship between Hox gene expression and limb patterning

    The removal of the AER results in truncated limbs where only the stylopod 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.

    FGF-soaked beads can mimic the AER

    Implanta

    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]

    Ectopic limb formation

    Transplantation of the AER to fl

    Transplantation of the AER to flank mesoderm between the normal limb buds results in ectopic limbs. If the AER is transplanted closer to the forelimb bud, the ectopic limb develops like a forelimb. If the AER is transplanted closer to the hindlimb bud, the ectopic limb develops like a hindlimb.[23] If the AER is transplanted near the middle, the ectopic limb has both forelimb and hindlimb features.[24]

    AER does not specify limb identity

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