Fig. 2

Cyclopamine targets both PGCs and somatic cells to effect PGC mislocalization. (A) Schematic representation of the transplantation procedure used to differentiate cyclopamine action on PGCs and somatic tissues. Donor embryos were injected at the one-cell stage with EGFP-nanos1-3′UTR mRNA to selectively label PGCs and Alexa Fluor 594-dextran to label all donor cells, and one to three PGCs were transplanted from shield-stage (6 hpf) donors into unlabeled sphere-stage (4 hpf) hosts. Donor and host embryos were treated with 100 μM cyclopamine or an ethanol vehicle control prior to transplantation, and cultured until 24 hpf in cyclopamine-free medium. EGFP-expressing PGCs in the host embryos were then scored at 24 hpf for their localization to the presumptive gonad site. (B–E) Bright-field images, (B′–E′) EGFP fluorescence images, and (B″–E″) Alexa Fluor 594 fluorescence images of representative host embryos resulting from the following donor/host treatment regimens: (B) ethanol-treated donor and host; (C) cyclopamine-treated donor and host; (D) cyclopamine-treated donor and ethanol-treated host; and (E) ethanol-treated donor and cyclopamine-treated host. (F) Quantification of PGC mislocalization, scoring at least 100 transplanted PGCs for each donor/host condition per experiment. Data represent the average of three or more experiments, with error bars representing the standard error of the mean. (G–L) Distribution of sdf1a transcripts in ethanol- (G–I) and cyclopamine-treated (J–L) embryos from gastrulation to mid-somitogenesis. Embryos at the shield (G and J), 3-somite (H and K), and 15-somite (I and L) stages are shown. Scale bars: 200 μm.