Fig. 3. Krm2 and LRP6 are required for NC induction in Xenopus. (A-D) Morpholino-mediated knockdown of krm2 expression. (A) Embryos at the eight-cell stage were injected animally with 10 ng Krm2 MO-1 plus either 0.8 ng PPL (middle panel) or V5-Xkrm2 mRNA (right panel), respectively. Control, PPL mRNA injected. Normal or hyper-pigmentation was observed in 9% (n=35) of (Krm2 MO-1 + PPL) and in 44% (n=27) of (Krm2 MO-1 + V5-Xkrm2) mRNA injected embryos, respectively. (B) Embryos at the eight-cell stage were injected animally with 10 ng Krm2 MO-1 or 20 ng Krm2 MO-2 or control MO (CoMO), respectively. Shown are whole-mount in situ hybridizations for slug expression at neurula stage in anterior view. ß-gal lineage tracer is stained in red. (C) Statistical overview of MO injection experiment in B. (D) Top left: diagram of experiment. Two-cell-stage embryos were injected animally with 7.5 ng CoMO or Krm2 MO-1, and animal caps (ACs) were explanted at stage 8-9 and combined with dorsolateral marginal zones (DLMZs) of uninjected or Krm2 MO-1 (7.5 ng) injected gastrula-stage embryos. Conjugates were assayed at stage 20 for slug expression by in situ hybridization. Top right: conjugates of DLMZs and CoMO-injected caps. Arrowheads indicate slug expression. Inset: ACs injected with CoMO and processed for slug expression. Bottom left: conjugates of ACs and Krm2 MO-1 injected DLMZs. Arrowheads indicate slug expression. Bottom right: conjugates of DLMZs and Krm2 MO-1 injected ACs. (E,F) Phenotypic analysis of LRP6 MO in X. laevis (E) and X. tropicalis (F). All embryos were injected equatorially at the two-cell stage. (E) (a) Injection of 5 ng CoMO. (b,c) Co-injection of 5 ng LRP6 MO and either 1 ng control (PPL) (b) or 400-600 pg human LRP6 mRNA (c). (e) Statistical overview of a-c. (d) Injection of 20 pg dkk1 mRNA. (F) Xenopus tropicalis embryos injected with 1.25 ng CoMO or LRP6 MO show the displayed phenotypes at frequencies of 89%, n=38, upper panel and 98%, n=44, lower panel. (G) Two-cell-stage X. laevis embryos were injected equatorially in one blastomere with 5 ng CoMO or increasing LRP6 MO doses as indicated. Neurula stage embryos were processed for slug expression by in situ hybridization and are shown in anterior view. ß-gal lineage tracer is stained in red.