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Ten microliters of each RT-PCR reaction were electrophoresed on a 2.5% agarose gel with 0.1 |xg/ml ethidium bromide in TAE 1X buffer. Gels were visualized by UV transillumination and photographed with a DS-34 Polaroid camera (Polaroid, Waltham, MA). The size of the amplified products was 310 bp for Sox9 and 243 for p-actin. The intensity of each band was quantified by densitometry using the Scion Image program. Results were expressed as arbitrary units of the ratio between Sox9 and p-actin mRNA levels. In Situ Hybridization Bar = 2.0 ^m. In embryos at 12.5 and 13.5 dpp, the gonads were processed in situ attached to the dorsal body wall. At 18.5 dpc and 30 dpp, the gonads were dissected out and processed. A total of 11, 15, 7, and 11 gonads at 12.5 dpc, 13.5 dpc, 18.5 dpc, and 30 dpp, respectively, were processed. Whole-mount in situ hybridization for detection of Sox9 transcripts was carried out with probes kindly provided by Dr. Ramon Merino (Cantabria University, Spain). Reactions were performed in vitro using T3 or T7 RNA polymerase to generate sense and antisense RNA probes labeled with di-goxigenin-UTP. The complete procedure was as previously described by Wilkinson and Nieto. Reactions were developed with BCIP/NBT or with purple AP substrate (Boehringer-Mannheim, Roche, Indianapolis, IN). Statistical Analysis Profiles of Sox9 transcripts were quantified as mean ± standard deviation of at least three independent determinations. Variance between experiments was assessed by two-way ANOVA. The mean was used to evaluate the significant differences by paired Tukey test (P < 0.001).