JCS01738 Supplementary Material

Journal of Cell Science 118, 1449-1459 Published by The Company of Biologists 2005

doi:10.1242/jcs.01738

Materials and Methods

Whole-mount in situ hybridization

Whole mount in situ hybridization was performed as previously described by Kanai-Azuma et al. (Kanai-Azuma et al., 1999; Kanai-Azuma et al., 2002). In short, the genital ridges and their cultured explants were fixed in 4% PFA-PBS for 4 hours and then dehydrated in methanol. The samples were rehydrated, pretreated with 10 mg/ml proteinase K in PBST for 30 minutes, and then hybridized with digoxigenin (DIG)-labeled antisense Sry RNA probes (Bullejos and Koopman, 2001) (kindly provided by Peter Koopman, University of Queensland) in a solution containing 50% formamide, 10% dextran sulfate, 53 SSC, 1% SDS, 50 mg/ml heparin and 50 mg/ml denatured yeast RNA at 70°C for 16 hours. After treatment with RNase A (100 mg/ml; Sigma) at 37°C for 15 minutes, they were washed twice with 23 SSC/53SSC at 65°C for 1 hour. The signals were detected by an immunological method using alkalinephosphatase-conjugated anti-DIG antibody (Roche Molecular Biochemicals) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitro blue tetrazolium (NBT) (Roche) as the chromogen.

References

Kanai-Azuma, M., Kanai, Y., Okamoto, M., Hayashi, Y., Yonekawa, H. and Yazaki, K. (1999). Nrk: a murine X-linked NIK (Nck-interacting kinase)-related kinase gene expressed in skeletal muscle. Mech. Dev. 89, 155-159.

Kanai-Azuma, M., Kanai, Y., Gad, J.M., Tajima, Y., Taya, C., Kurohmaru, M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P.P.L. and Hayashi, Y. (2002). Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129, 2367-2379.

Files in this Data Supplement:

Supplemental Figure 1 -

Fig. S1. Changes in relative number of PAS-positive cells per unit area in each region (I-V) during early phases of testis differentiation. Vertical axis represents the relative number of PAS-positive cells per unit area [total cell number in each region (Fig. 4C) divided by the total area in each region; cell number per mm²], while horizontal axis represents the regions I-V of the gonads. In each graph, bars of the same color show the cell number in each region of the same gonad (14 ts, five gonads; 15 and 16 ts, each four gonads).

Supplemental Figure 2 -

Fig. S2. Whole mount in situ hybridization showing Sry expressions in developing XY genital ridges in vitro. Sry expression is first detected in the central region of XY gonads at 12 ts (most left plates; ‘0h’). In XY explants isolated at 12 ts, Sry expression is increased after being cultured for 6 hours, and then its expression is rapidly reduced to the undetectable level until 12 hours. In XY explants isolated at 10 ts, Sry expression is properly induced in the central region of the gonadal area, and then its expression domain is expanded to their whole gonadal area until 12 hours, although their expression levels appear to be lower than those of the cultured explants isolated at 12 ts. Anterior is shown on the left in each plate, while anterior and posterior edges of the gonadal area are indicated by arrowheads.

Supplemental Figure 3 -

Fig. S3. Phosphorylation levels of ERK (a downstream effector of MEK) in the genital ridges treated with MEK inhibitor PD98059. Immunoblot analyses of phosphorylation levels of ERK in XY and XX genital ridges (10-11 ts) cultured for 24 hours in the presence or absence of PD98059 (50 mM). The phosphorylation levels of ERK are clearly reduced by addition of PD98059 in both XY and XX explants. Repeat experiments were performed three times, and similar results were obtained each time.