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.

Isolation was conducted by designing PCR primers that correspond to sequences of Shh that are conserved in Drosophila and mouse and involved in limb bud formation.
 The clone was then used as a template  to screen a cDNA library from stage 22 limb bud RNA.
The group ectopically expressed the gene by taking advantage of a retroviral vector to insert the cDNA into chick cells.
 There are unique types of this retroviral vector that only infect specific strains of avian species.
 Therefore, this group used a retroviral vector termed RCAS-E, which lacks a type E envelope protein, and is able to infect certain chick embryo fibroblasts with Shh.

Results showed digit duplications, with the most common being 4-3-3-4, with digit 2 missing.
Though there was variability, it was clearly consistent with anterior to posterior positional patterning.
 Variations   were due to the amount of tissue grafted, and the location of the graft.
 These findings indicate that Shh could substitute  for the function of the ZPA.
Thus Shh is sufficient for ZPA action.
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.
Isolation was conducted by designing PCR primers that correspond to sequences of Shh that are conserved in Drosophila and mouse and involved in limb bud formation.  The clone was then used as a template  to screen a cDNA library from stage 22 limb bud RNA. The group ectopically expressed the gene by taking advantage of a retroviral vector to insert the cDNA into chick cells.  There are unique types of this retroviral vector that only infect specific strains of avian species.  Therefore, this group used a retroviral vector termed RCAS-E, which lacks a type E envelope protein, and is able to infect certain chick embryo fibroblasts with Shh.
Results showed digit duplications, with the most common being 4-3-3-4, with digit 2 missing. Though there was variability, it was clearly consistent with anterior to posterior positional patterning.  Variations   were due to the amount of tissue grafted, and the location of the graft.  These findings indicate that Shh could substitute  for the function of the ZPA. Thus Shh is sufficient for ZPA action.
Zone of polarizing activity