Fig. 3. Inhibition of Shh signaling by DHCR7. (A) DHCR7 mRNA (2 ng) was microinjected unilaterally into one blastomere of the two-cell stage embryo. (B) Two-cell stage embryos were injected with 250 pg of Xenopus Shh mRNA (middle row), or together with 500 pg of DHCR7 mRNA (bottom row; Shh + DHCR7). Embryos were subjected to whole mount in situ hybridization at stage 35 (tailbud) using indicated probes. When Shh mRNA was co-injected with DHCR7 mRNA, marker gene expression was restored to the wild-type level (top row). The rescue efficiency was: Pax6, 87% (n=46); Pax2, 90% (n=48); Gli1, 91% (n=34); Patched1, 93% (n=28). (C) RT-PCR analysis of RNA isolated from animals caps injected with Chordin (50 pg) plus Shh (100 pg) (lane 2), Chordin plus Shh with 1 ng of DHCR7 (lane 3), Chordin plus 100 pg of Shh-N (lane 4) and Chordin plus Shh-N with 1 ng of DHCR7 (lane 7) mRNA. Uninjected stage 28 equivalent animal cap control (lane 1). (D) Gli-reporter assays using animal cap assay. (E) Schema of a conjugation experiment. (F) Gli-reporter gene assay using animal cap conjugates.