C: This figure is a visual representation of the most common digit phenotype resulting from grafting Sonic hedgehog-RCAS-infected CEF (chicken embryo fibroblast) cells onto the anterior margin of a limb bud. The resulting digit pattern is a mirror-image duplication of digits 4 and 3 with digit 2 missing, forming a sequence of 4-3-3-4. In many such cases, the two central digits (the two 3s) appear fused, showing the effect of ectopic Sonic hedgehog expression on limb development and digit formation. This digit pattern indicates that the Sonic hedgehog-expressing cells have the ability to mimic some aspects of ZPA (zone of polarizing activity) grafts, which are known to induce mirror-image duplications of limb structures when grafted to the anterior margin of a limb bud.

D: This figure illustrates the outcome when grafts of Sonic hedgehog-expressing cells are placed in the posterior region of limb buds, instead of the anterior margin. Unlike the anterior grafts, these posterior grafts do not result in changes in the number of digits, which is consistent with the idea that the polarizing activity is already present in the posterior region of the limb bud. However, some such grafts did produce distortions in the shape of limb elements. Most commonly, a slight posterior curvature in the distal tips of digits 3 and 4 was observed when compared with wild-type wings. This outcome indicates that while the Sonic hedgehog-expressing cells can alter the shape of limb elements, their ability to induce mirror-image duplications of digits is limited to the anterior region of the limb bud.

Explanation of the results:

1. Panel A: This panel shows the expression of Shh in the chick limb bud with the anterior (A) and posterior (P) axis labeled. The black staining indicates the presence of Shh, which is expressed in the posterior region of the limb bud, known as the zone of polarizing activity (ZPA). This region is crucial for the proper specification of digit identities along the anteroposterior axis.
2. Panel B: This panel serves as an unimplanted control limb, likely displaying normal limb development and patterning. It represents the standard formation of digits and long bones (humerus, radius, and ulna) in the absence of experimental manipulation.
3. Panel C: In this panel, Shh-expressing cells are implanted in the anterior margin of the limb bud. The ectopic expression of Shh in the anterior region results in the disruption of the normal anteroposterior patterning. Consequently, the formation of digits and long bones is affected, leading to duplicated or altered digit patterns.
4. Panel D: Here, Shh-expressing cells are implanted in the posterior margin of the limb bud, where Shh is naturally expressed. This may lead to enhanced or altered anteroposterior patterning, depending on the concentration of Shh signaling in the region. The additional Shh signaling might strengthen the existing gradient, potentially causing changes in the digit identities or the overall limb morphology.
5. Panels E and F: These panels indicate control and infected limb buds, respectively, with a retrovirus expressing Shh. In Panel E, the control limb bud does not have any Shh ectopic expression and represents normal Hoxd-13 expression levels. Panel F displays the infected limb bud, where Shh expression has been induced in the anterior region. The upregulation of Hoxd-13, a homeotic gene involved in limb development, suggests that Shh signaling influences Hoxd-13 expression, thereby affecting limb patterning.

How Shh controls anteroposterior patterning in limbs:

Shh is a key signaling molecule in the development of the vertebrate limb, especially in the determination of the anteroposterior axis. Shh is produced in the ZPA, a group of cells located at the posterior margin of the developing limb bud. This region is essential for the proper specification of digit identities along the anteroposterior axis.

1. Gradients of Shh signaling: Shh forms a concentration gradient across the limb bud, with the highest levels of expression in the ZPA and progressively lower levels towards the anterior margin. Cells in the developing limb bud respond to the different concentrations of Shh, leading to the specification of distinct digit identities.
2. Interaction with other signaling pathways: The establishment of the anteroposterior axis in limb development is a highly coordinated process that involves cross-talk between several signaling pathways, including the BMP and FGF pathways. Shh signaling interacts with these pathways to ensure proper limb patterning.
3. Regulation of Hox genes: Shh signaling regulates the expression of Hox genes, such as Hoxd-13, which are critical for proper limb development. These genes encode transcription factors that determine the identities of the different limb elements, including the digits and long bones, along the anteroposterior axis.
4. Feedback loops and the progress zone model: The developing limb bud also contains a region called the progress zone, where undifferentiated cells are exposed to FGF signaling from the apical ectodermal ridge (AER). Shh signaling from the ZPA maintains the expression of FGFs in the AER, which in turn maintains Shh expression in the ZPA. This feedback loop between Shh and FGF signaling contributes to the regulation of limb bud outgrowth and the establishment of the anteroposterior axis.

In summary, Shh signaling plays a crucial role in determining the anteroposterior patterning of the vertebrate limb by establishing concentration gradients, interacting with other signaling pathways, regulating Hox gene expression, and participating in feedback loops with other signaling molecules.