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Soon after, it was shown that beads soaked in the amino-terminal cleavage product of Shh (Shh-N) could also produce digit duplications in the chick wing (16). Beads soaked in Shh-N were used to characterize signaling in the chick wing, and this confirmed similarities with polarizing region signaling (17). Thus, beads soaked in different concentrations of Shh implanted at the anterior margin of chick wing buds led to dose-dependent changes in digit pattern (Fig. 1C). There Shh Application to Limb 25 Fig.

Pepinsky, R. , Day, E. , et al. (2000) Mapping Sonic hedgehogreceptor interactions by steric interference. J. Biol. Chem. 275, 10,995–11,001. 2 Application of Sonic Hedgehog to the Developing Chick Limb Eva Tiecke and Cheryll Tickle Abstract Here, we describe methods for applying Sonic hedgehog (Shh) to developing chick limbs. The Sonic hedgehog gene is expressed in the polarizing region, a signaling region at the posterior margin of the limb bud and application of Shh-expressing cells or Shh protein to early limb buds mimics polarizing region signaling.

3. Microinjection of Synthetic mRNA into Xenopus Embryos Depending on the construct (gene of interest), 1 or up to 2000 pg in vitro synthesized capped mRNA can be microinjected into blastomeres of 1- to 32-cell stage embryos in 5 to 10 nL H2O. Fate maps can be found in Refs. (12–14). The correct injection volume needs to be adjusted under a microscope with the help of an eyepiece micrometer. To do so, measure the diameter of the drop and calculate the respective volume (v = 4/3Ur3). After injection, the embryos are transferred into 1× MBS and kept at 15°C.

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