Fig. 1. CXCR4 is expressed in PGCs during and after colonization of the gonad. (A) Probes prepared from E10.5 (two biological replicates) and E12.5 (two biological replicates) PGCs were applied to Affymetrix chips. Chip data was analyzed using MicroArray Suite v5.0 software (Affymetrix) to generate signal intensity data, and to statistically determine presence and absence calls. The average chip signal was normalized to an arbitrary value of 1000. The bar graph shows the signal intensity of the CXCR4 probe set for each sample. CXCR4 was called present in all samples. The PGC marker gene Kit was also called present in all samples. The somatic marker gene Steel (Kitl - Mouse Genome Informatics) was called absent. (B) CXCR4 expression was confirmed by RT-PCR. Lane 1, E10.5 PGC cDNA; Lane 2, RT-; Lane 3, H₂O blank; Lane 4, E12.5 PGC cDNA; Lane 5, E10.5 whole-embryo cDNA. (C) Cxcr4 message is enriched in PGCs relative to the somatic tissue. The level of Cxcr4 transcripts in E11.5 PGCs (GFP+) or somatic tissue (GFP-) were quantified by SYBR-green based RT-PCR. The meiotic marker, STAG3 (Pezzi et al., 2000) was used as a positive control for PGCs, and the gonadal markers SPARC and cystatin C (CST3) (Wertz and Herrmann, 2000) were used as positive controls for the somatic component of the gonad. For CXCR4 and STAG3, the PGC cDNA was used to generate a standard curve and expression in this tissue was set to an arbitrary value of 100. For SPARC and CST3, the somatic tissue cDNA was used to generate standard curves. Expression was normalized to ODC levels in the somatic and PGC samples. (D) SDF1 is expressed in the genital ridge area. Anti-SDF1 staining is shown in red. This slice was taken from an E9.5 +/Oct4PE:GFP⁺ embryo. A BSA-coated bead was placed in the aorta and the slice was cultured for 12 hours, then fixed and stained with 2.5 µg/ml anti-SDF1 antibody. Arrows indicate the region of more intense SDF1 staining in the floor plate of the neural tube, and arrowheads indicate the mesonephros and adjacent mesenchyme. The germ cells are marked by bright green GFP fluorescence. (E) Control for SDF1 staining. This control slice was treated in the same way as the experimental slice in D, but the anti-SDF1 antibody was omitted. Scale bar in D: 82 µm for D,E.

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