Zone of polarizing activity

The zone of polarizing activity (ZPA) is an area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis. Limb bud is undifferentiated mesenchyme enclosed by an ectoderm covering. Eventually, the limb bud develops into bones, tendons, muscles and joints. Limb bud development relies not only on the ZPA, but also many different genes, signals, and a unique region of ectoderm called the apical ectodermal ridge (AER). Research by Saunders and Gasseling in 1948 identified the AER and its subsequent involvement in proximal distal outgrowth. Twenty years later, the same group did transplantation studies in chick limb bud and identified the ZPA. It wasn’t until 1993 that Todt and Fallon showed that the AER and ZPA are dependent on each other. The zone of polarizing activity (ZPA) is an area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis. Limb bud is undifferentiated mesenchyme enclosed by an ectoderm covering. Eventually, the limb bud develops into bones, tendons, muscles and joints. Limb bud development relies not only on the ZPA, but also many different genes, signals, and a unique region of ectoderm called the apical ectodermal ridge (AER). Research by Saunders and Gasseling in 1948 identified the AER and its subsequent involvement in proximal distal outgrowth. Twenty years later, the same group did transplantation studies in chick limb bud and identified the ZPA. It wasn’t until 1993 that Todt and Fallon showed that the AER and ZPA are dependent on each other. Patterning along the limb bud requires signals from many sources. Specifically, proteins called transcription factors (TF) help control the rate at which a gene is transcribed. The limb bud expresses a TF called ALX4 at the anterior part of the mesoderm, with the TF HOXB8 being expressed at the posterior portion. The Alx4 region, the medial region, and the Hox8 expressing area meet at a proximal area where the AER develops. The ZPA forms where the Hox8 region joins the AER. These regions are dependent on signaling in order for the appropriate induction events to occur. The AER expresses FGF8 which induces Shh expression in the posterior mesoderm. Shh then stimulates FGF4 to be expressed in the posterior part of the AER. After these events, there is a co-dependence between FGF-4 and Shh for their subsequent expression and maintenance. Additionally, Wnt7a is expressed in the dorsal ectoderm, is needed to maintain the FGF and Shh signaling. Saunders and Gasseling published data in the Journal of Experimental Biology in 1948, showing that reference marks inserted near the rim of the apical border of the wing bud are dispersed throughout the whole forearm of the wing. This led them to believe that the apical ectoderm may play a role in forming parts of the wing. To test this, they removed apical ectoderm from wing buds which yielded deformed wings. When they removed dorsal ectoderm, normal wings formed. These results showed that the cells of the apical ectoderm have a precise fate to form specific regions of the wing. In 1968, Saunders and Gasseling did transplantation studies using tissue from chick limb bud. Removing cells from the posterior region of the limb, they transplanted them to the anterior region and noticed that extra digits formed in the anterior area and these digits were mirror images to the normal ones. This posterior mesenchyme was the ZPA, which is now known to express the protein sonic hedgehog (Shh). One hypothesis is that at high concentrations, this unknown morphogen causes mesenchyme to form on the posterior side, while low concentrations induces meshenchyme to form on the anterior end. Identifying this morphogen was the next crucial step.The first hypothesis came from Tickle et al. who showed that when retinoic acid is placed in the anterior margin of the limb bud, mirror image duplications result. However, concentrations of retinoic acid that cause mirror image duplications induce high levels of a downstream gene, retinoic acid receptor Beta, which is not seen in the posterior region. It is now known that endogenous retinoic acid acts permissively prior to limb bud initiation to allow the budding process to begin, and that the specific morphogen, hypothesized to be Shh, is normally expressed independently of retinoic acid in the posterior region of the limb bud. By looking at signaling homologs of other organisms, the segmentation gene of Drosophila, hedgehog, served as a viable candidate. The idea that Shh is required for proper ZPA signaling and anterior/posterior limb formation needed to be tested. Riddle et al. took Saunders and Gasselings findings to the next step and proved that Shh is the morphogen within the ZPA that is required for anterior posterior patterning. By isolating the Shh gene and implanting it into the anterior limb bud, mirror image digit duplications formed.